Science topic

Signal Transduction - Science topic

The intracellular transfer of information (biological activation/inhibition) through a signal pathway. In each signal transduction system, an activation/inhibition signal from a biologically active molecule (hormone, neurotransmitter) is mediated via the coupling of a receptor/enzyme to a second messenger system or to an ion channel. Signal transduction plays an important role in activating cellular functions, cell differentiation, and cell proliferation. Examples of signal transduction systems are the GAMMA-AMINOBUTYRIC ACID-postsynaptic receptor-calcium ion channel system, the receptor-mediated T-cell activation pathway, and the receptor-mediated activation of phospholipases. Those coupled to membrane depolarization or intracellular release of calcium include the receptor-mediated activation of cytotoxic functions in granulocytes and the synaptic potentiation of protein kinase activation. Some signal transduction pathways may be part of larger signal transduction pathways; for example, protein kinase activation is part of the platelet activation signal pathway.
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The Wnt signal transduction cascade controls myriad biological phenomena throughout the development and adult life of all animals.
I have seen many research works that have been used LiCl as a WNT activator but few research studies attempted to use it as an inhibitor.
Could anyone clarify, how LiCl would be used as a WNT inhibitor in developmental biology research?
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Saurabh Mandal thank you very much for your descriptive answer.
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Two signaling pathways can activate the host innate immunity against viral infection. One of the pathways utilizes members of the Toll-like receptor (TLR) family to detect viruses that enter the endosome through endocytosis. The TLR pathway induces interferon production through several signaling proteins that ultimately lead to the activation of the transcription factors NF-κB, IRF3 and IRF7. The other antiviral pathway uses the RNA helicase RIG-I as the receptor for intracellular viral double-stranded RNA. RIG-I activates NF-κB and IRFs through the recently identified adaptor protein MAVS, a CARD domain containing protein that resides in the mitochondrial membrane. MAVS is essential for antiviral innate immunity. 
Is there any references that explain the involvement of RIG-I mediated antiviral innate immunity induced by Baculovirus?  
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Hi, did it implies that double stranded DNA virus could also induce the production of type I IFN through TLR-mediated or RIG-I-mediated pathways, where the IRF3/7 was recruited? thx
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I'm trying to prepare biological network pathways and signal transduction. Based on the requirement, please suggest online available tools to draw scientific figures.
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I'd suggest Biorender
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I am trying to detect phosphorylated forms of Akt (Ser and Thr) in rat skin tissue. I have used p-Akt (S473) (587F11) Mouse mAb #4051 from Cell Signaling, and although this one works flawlessly in human, it does detect two bands in rat samples, and the upper one does not run at 60kDa, but more like at 50-55kDa). The similar is observed with Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 and Phospho-Akt (Thr308) (C31E5E) Rabbit mAb #2965.
I include the sample of stimulated p-Akt in human cells versus one in rat tissue using the same Ab.
If you validated any p-Akt Ab that worked for your stimulated/unstimulated rat tissue, and does not detec the bottom band, please let me know, since it would save me a lot of time and $$$.
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@Edita
Were you able to get expression of pAkt?
I want to show the expression by Flowcytometry. I am struggling with stimulation step.
May you help me with stimulation step so that I may go ahead.
Thanks in advance.
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Plants acquire nutrients from soil but we also know that there is variability in soil fertility even within same field. However, the area where nitrogen availability is less plant always tend to spread its root towards the area where sufficient N is available. So my question is that what make plant roots to spread in search of N? How plant comes to know that N is available in that specific direction? is there any signal transduction by roots to find N rich area? if yes then kindly share its mechanism.
Regards
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There is not a "mechanism" that we are aware of that lets plants know where nutrients are in the soil. Nutrients reach plant roots by one of 3 ways, diffusion of nutrients (a slow random dispersion of molecules), root interception (the search party finds nutrients) or mass flow (nutrients moving with water).
Roots grow and find nutrients. When they find nutrients they become more dense in that area until the nutrients are depleted. Yes, if the nutrients aren't there then it is wasted energy. If doesn't find enough nutrients then all of the wasted energy results in the plants death.
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Is the introduction of labeling bad while detecting small molecules? If yes, what are the major disadvantages of using labels for signal amplification in the detection of small molecular weight ( <1 kDa ) compounds?
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For a given size label, the effect on molecular properties will depend on the size of the molecule you want to label. This is even true for isotope effects, they are much stronger for 2H/1H than for 13C/12C. In addition, for a small molecule the chances that the label sterically interferes with binding of the molecule into the pocket of its receptor are much larger than for, say, a big protein.
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because all steps need ATP. while prolonging the process, more ATP will be used.
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I think MAP-kinases can regulate multiple pathways and different downstream kinases have specificity to activate different pathways differentially depending on the cellular availability of substrates. A cascade also allows differential inhibition of each component. These are like safety valves in the system at multiple places in the circuit to have better control of the cellular system.
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I want to study cell signaling. where the protein synthesized from signal transduction by cell A is secreted and is important for signal transduction pathway to cell B. Plz suggest such 2 cell lines, where I can track its live progress.
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Something like interferon secretion sounds like it is what you are looking for. You can find some cells (e.g. PMDC05) that will secrete IFN upon TLR stimulation....and then most cells will respond to IFN where you will see many interferon stimulated genes
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I went through papers in which people had carried out thymidine block followed by nocodazole block for synchronizing cells in the G2-M phase. However, after the thymidine block for 24 hrs,a 3 hr gap is maintained before the nocodzole tretment.
What would be the consequence if this 3 hr duration is not followed and instead cells are treated with nocodazole immediately after the 24 thymidine treatment?
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rounding of the cells and loss of attachment is normal cell behavior that precedes the cell division and since the cells are stuck in M phase they round and await the division.
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I need an updated (published in the last 5 years) book clearly and extensively explaining the concepts underlying developmental processes (embryogenesis, flower, root, shoot and leaf development) in plants and how plant endogenous signals and environmental signals are perceived and integrated into the initiation, proceeding and control of developmental processes.
Thanks in advance
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Mr. Hicham Mechqoq, thanks for your answer, it has been really useful.
Dr. Maged G. Bin-Saad, the most recent publication with the title you wrote dates back to 1987, I was looking for recent ones.
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Hi,
I'm currently having problems with Western blots using the following antibodies from Cell Signaling:
Phospho-Stat3 (Tyr705)
STAT3 (total)
Phospho-Stat5 (Tyr694)
STAT5 (total)
They seem to work very inconsistently, sometimes giving no signal at all. They used to work better for the first 6 months, giving the correct bands. It's not a technical issue - I'm regularly running multiple blots with ~30 different primary Abs, and they work consistently. Ponceau and GAPDH indicate the presence of protein. It's also not an issue of molecular weight - other 100-200 kDa proteins are detected without problems.
I would like to try out some new antibody clones, which ones would you recommend?
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I will suggest WB STAT5 for better result
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I have grown HepG2 cells for a couple of years, never had any problems serum starving and stimulating the cells (using pS473-Akt as a readout). But recently my cells stopped to respond: either pS473-Akt level is still high in starvation condition or still low after stimulation. I've changed media, serum, tested out different batches of cells I froze before which were definitely working back then but still can't solve the problem. One thing I want to mention is, once I changed to a new media and the cells became good but right after one split, they were bad again! Anyone has experienced the same thing?
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The mycoplasm contamination can be a problem in cell signaling pathways.
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We have compounds that have demonstrated innate immune stimulation in vivo, however are unclear on the mechanism of action/specific receptor involved. We had thought they stimulated TLR9, however upon analysis using a TLR9-specific reporter cell line, we found this not to be the case. Since we don't know where exactly to look next and creating/buying receptor-specific cell lines is time-consuming/costly, we are trying to devise a cheap and quick method.
We believe it isn’t any of the other TLRs, so I devised a quick way to rule this out involving measuring MyD88 & TRIF levels in stimulated cell lysates via western blot, as I believe one out of these two molecules is always required for TLR signal transduction – no increase in signal over the negative control in each would therefore show that the compounds are operating via a TLR-independent mechanism. However, I am unaware whether stimulation of the associated TLR causes increased expression of these molecules, or simply localisation to the receptor site, and cannot find any examples of this technique used to measure TLR stimulation in the literature. Essentially – do you think this would work before I order in antibodies? Or if not, are there any other cheap, quick methods for measuring stimulation of all TLRs?
Thank you.
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Interesting question. Unfortunately, I dont think that monitoring expression levels of these molecules will give any sensible indication for pathway activation. It is mostly pottranslational modifications which change upon receptor activation.
You mentioned that these compounds are immune stimulators in vivo. Do you think that they themselves are stimulating cells or are they potentiating TLR ligands or other cytokine-induced signaling? Are these cell-permeable molecules? MAPK activation is a nice indication of cell stimulation (as suggested by Vadim), but will not reveal the identity of the receptors involved. However, monitoring MAPK / NFkB activation upon treating highly sensitive cells with the molecules +/- TLR ligands will be a nice start! Good Luck, Manoj.
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I am using calcium channel blockers to reduce intracellular calcium level.
Based on previous reference we informed that intracellular calcium amout is 100nM.
So after treatment of CCBs which percentage inhibition of calcium level disrupt calcium homeostasis as well as which percentage of calcium inhibition can effect on intracellular signal transductions?
Thanks in advance.
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I have a bit of a problem understanding your question.
I think it is very difficult to lower the basal Ca2+ level in the cytosol (100 nM) by using inhibitors of Ca2+ channels, because the basal level of Ca2+ (100 nM) in controlled by Ca2+-ATPases.
One way to go could be to permeabilise the membrane and control the extracellular concentration of Ca2+ in the medium. But it of course depend on what you want to study.
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I start this discussion so we can all share our favourites books on cell signaling or signal transduction. I am highly interested in GPCRs.
So I will start first.
"G Protein-Coupled Receptors: Structure, Signaling, and Physiology" by Sandra Siehler, Graeme Milligan is my favourite book so far.
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For a deeper/broader understanding, I suggest you start with:
Cellular Signal Processing: An Introduction to the Molecular Mechanisms of Signal Transduction 2nd Edition
by Friedrich Marks, Ursula Klingmüller, Karin Müller-Decker
I like its approach to cellular signaling and it shows how GPCRs fit into the
signal processing apparatus of the cell.
Then you can dive into:
G Protein-coupled Receptors: Molecular Pharmacology
by Georges Vauquelin, Bengt von Mentzer
None of the books capture the latest developments in GPCR biology as
the field is moving very fast. For this you have reviews by experts.
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What is the simplest, quickest, and/or cheapest way of finding out whether a molecule is involved in a novel quorum sensing / cell-cell signaling / signal transduction pathway?
Most literature explains assays that build on systems that have already been established, involving AHLs, AI-2, DKPs, DSFs, HAQs, etc., but our suspected signal molecule is not similar to those. It might be involved in cessation of cell growth, and we sort of have growth curves at different concentrations of the compound.
Outside of forward and reverse genetics, which would be especially difficult for our organism, an Archaeal methanogen, I’ve seen the most plausible options in this paper:
Affinity chromatography and photo-affinity labeling don’t seem simple or quick or cheap, though.
I’d appreciate any suggestions.
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Dear Elisa,
It is always difficult to go from growth experiments to the identification of an active compound, especially if your model organism is hard and slow to grow, you lack a fast and simple assay for the activity, and if you are not based in a biochemical lab. However, there are some simple experiments that you can do to narrow down the nature of your active compound.
I would suggest that you try some simple growth addition experiments to demonstrate that the effect is real, and that cells are responding to this compound in a dosage-dependent manner. The easiest experiment is to add some filter-sterilised (FS) culture supernatant from cultures in which you see cell death or growth-inhibition to a fresh culture - you would expect to be able to inhibit the growth of this or see cell death occurring earlier than if you did not add FS culture supernatant.
If this works, then there are some simple tests you can do : test FS culture supernatants from cultures grown in different media, grown for different lengths of time, and grown to different cell densities. Try fractionating your FS culture supernatant using dialysis membrane or filters (with different pore sizes) to see if you can put a size limit on the active compound; try heat inactivating the compound, and precipitating or extracting it with alcohol, phenol, ammonium sulphate, etc.
The aim of all of this is to produce a semi-purified concentrated extract that you could then consider fractionating by HPLC and then identifying the compound by mass spectroscopy (if you had access tho this type of analytical equipment). Being able to demonstrate that you have a semi-purified extract plus a simple phenotype you can assay activity with would be a good end-point for a project where you could not do any further biochemical analyses.
Regards, Andrew.
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bistability is about the switching between two states from one state to another state. it correlates with Michaelis-Menten equation that explain enzyme kinetic activity. Anyway, there are some theoretical studies showed that it can be applied in phosphorylation. Could it possible to  applied to other signal transductions? Thank for your help 
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Dear Myself
Since I studied 5 years ago. There are many theories could be applied for signal transduction pathway, i.e. Michaelis-Menten kinetics. It is possible for using MM to explain bistability for the different signal transduction pathways.
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I would like to know the difference between newborn and adult cardiac fibroblasts inculdes characters and signal transduction. Who can suggest some references? Thank you.
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Does anyone know if the IL2R gamma subunit is absolutely required for signaling, especially on neutrophils, or if there is signal transduction in the absence of IL2Rg to some extent?
Thank you.
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I believe the beta chain has a JAK site, so it can also induce an inter-cellular response.
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Steroid hormones are usually transported in the blood stream by carrier proteins. At their target cells they are released and can then either bind to surface receptors or diffuse through the plasma membrane due to their lipophilic nature. In the cell they are bound to receptors that enter the nucleus and influence gene expression. Some aspects of this model are not clear to me:
1.) How do carrier proteins in the bloodstream "know" when to release the hormones? How are target cells recognized? The ability of steroid hormones to directly pass the plasma membrane makes specificity somewhat difficult to achieve.
2.) So most steroids can pass through the plasma membrane because they are lipophilic. But then they have to travel through the hydrophilic cytoplasm to reach their destination. Are steroid hormones amphiphilic? Or are they immediately bound to a receptor protein once they pass the plasma membrane?
3.) If steroid hormones are capable to pass the plasma membrane they should also be able to pass the nuclear membrane without being bound to a receptor protein, yet this does not seem to be the case. The lipid composition of plasma and nuclear membrane is different, but can this explain the differential diffusion behaviour of steroid hormones?
4.) How are steroid hormones that have served their purpose removed from the cell? Are there deactivating enzymes? If yes, how are these regulated? Or can the steroid hormones, once released from their receptors, simply diffuse out of the cell? If this is the case, how is a "re-binding" of the hormone to another receptor protein prevented?
I would appreciate if somebody could help me with these questions and/or point me to literature that explains these processes in more detail.
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The mechanistically specific answers to your questions are complex and better answered by others. However, generally speaking:
1. Carrier proteins do not need to know when to release steroid hormones because the binding affinity between steroid hormones and their serum carriers is not very high. The off rates are probably high. When you think about the hypothalamic-pituitary-gonadal or hypothalamic-pituitary-adrenal axis, this makes sense. Serum cortisol or serum estrogen is more or less equally present throughout circulation. Its target effects are dictated by the presence of the receptors rather than the mobility of the ligand (since the ligand is everywhere).
2. The distance between the plasma membrane and various organelles looks quite large in a textbook, but this is for ease of depiction. In reality, many cells contain leaflets of ER and Golgi that are in close proximity to the PM and nuclear envelope. Moreover, in target tissues, the expression level of cognate receptors is relatively high such that there is a high probability that the steroid will collide with one of its receptors upon entering the cell.
3. I do not know if steroids can penetrate the nuclear envelope. Even if they could, they would not be able to influence transcription without being bound to their respective nuclear hormone receptors. It would not surprise me if their solubility in PM and nuclear envelope were different. All lipids are not created equally, and steroid hormones are significantly more hydrophilic than their parent cholesterol.
4. In most instances, steroids are cleared from circulation by dehydrogenases, reductases present in mitochondria and ER of the target tissue or elsewhere. Since the on/off rates of steroids are high, yes, there is continual diffusion in and out of cells. And as the serum levels of various steroids drop, eventually the intracellular level in target cells also drops. This is not only relevant for steroid degradation, but also for synthesis. For example: in early pregnancy, maternal cortisol is rapidly oxidized/inactivated by fetoplacental 11-beta-hydroxysteroid dehydrogenase 2 (11-HSD2). However, toward term, 11-HSD-1 expression increases in the chorioamnion, which reactivates cortisol and promotes a placental H-P-A feed-forward surge that ultimately triggers parturition. See this publication for more information:
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It is generally accepted that KRAS mutations shortcut the signal transductions which physiologically tranfer extracellular signals into nucleus. The mutated KRAS forms a blocking configuration at GTP-binding site, and becomes constitutively active. However, there are also some literatures reporting that KRAS mutated cells do not completely bypass upstream receptors. For example, in NSCLC EGFR knockdown inhibited cell proliferation in KRAS mutated cells-in a degree much more than it affected KRAS wild type cells. (BMC Med, 2012, 10: 28) There are several other papers indicating that KRAS mutated cells still need receptor tyrosine kinases to transduce signals. Does anyone have any idea about this contradiction? Is there any clinical evidence regarding this issue?
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I'm having a hard time finding a broad and in-depth review or book chapter that covers PDGF signaling pathway focusing on its relationship to ROS generation during signaling transduction in mammalian cells. Any suggestions?
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Thank you so much, Adil! It looks amazing!
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Studies I have found that use this tried and true technique (radioactive ligand binding assay) use cell culture preps or dissociated tissue. Do the cells need to be treated a certain way after FACS? Do you recommend membrane prep v intact cells? Thanks!
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I did some whole-cell (well acini, actually) binding studies back in the days. It worked nicely. Regarding flow-sorting prior to binding studies it shouldn't be a problem - just don't fix the cells at any point as it may induce configuration changes on surface proteins. The classic approach however would be a membrane prep. I guess you get more precise binding curves with membranes.
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For example, protein-protein/nucleic acid/lipid interaction pattern, signal transduction pathway they may participate, or some other else.
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Proline rich domains are involved in protein-protein interaction and signal transduction.
A typical motif XPpXP (where X is any aliphatic amino acid, P is always proline and p being sometimes proline) bind to SH3 domains, which are small domains found in several tyrosine kinases involved in signaling.
Some publications worth taking a look:
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As the question says, I am looking into the PathScan Antibody Array Kit from Cell Signaling and would like some opinions on it. Is the protocol difficult/confusing, are there better alternatives, and also if anyone has done it  with tissue samples.
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We performed the array with some types of cells (HepG2, macrophages, adipocytes) as well as adipose tissue. It was very easy and worked super. Unfortunately, the kit is not available anymore.
Cheers
Olga
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Hello
I have been getting great support from RG members.
Today, i want to ask about phosphorylation.
LATS1, which is known to be a critical component of Hippo pathway,
regulates YAP. As far as i know negative regulators of Hippo pathway
enhances nucleus localization of YAP by inhibiting LATS1.
Here is my question : LATS1 has phosphorylation sites.(S909 and T1079)
I studied that increased phosphorylation at such sites indicates elevated activity of LATS1, affecting loclization of YAP inside cells.
Is there any critical difference between the affect of dephosphorylation of
S909 and T1079 of LATS1? I have heard that phosphorylation or dephosphorylation at different sites in one same molecule can have different effects, which makes related research quite tricky and complex.
I want to know whether such differences exists in LATS1, as i just described above.(ex) Same YAP nucleus translocation but different target gene expression, etc.)   
Thank you for your interst and hope for your great achivements in your research
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Hi Jinkyu,
To my knowledge, the phosphorylation of both S909 and T1079 are required for the activation of LATS1. S909 is its autophosphorylation site, while T1079 is phosphorylated by upstream kinases MST1/2. The phosphorylation of LATS1 at T1079 by MST1/2 can recruit its activator MOB1, which in turn promotes LATS1 autophosphorylation at S909. Therefore, loss of phosphorylation at any site would influence the activity of LATS1, as well as YAP phosphorylation.
Hope this may help you. 
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Hello
(I will say my genes of interest as A,B and C)
Based on the results so far, A seems to induce translocation of B into nucleus from cytoplasm. As for B, B is already known as a transcriptional regulator of C.
For me, i hypothesized that A enhances nucleus localization of B, which finally upregulates mRNA level of C and finally protein level of C.
Here is my dilemma. I am doing experiments described above under high cell confluency, but I cannot see positive regulation of C by B under such condition. (B localization depends on cell confluency, so B is mainly located in cytoplasm in dense cell condition while it is mainly found in nucleus when cells are cultured under sparse confluency)
I think that  because transcription activity of B is very low in dense cell condition, it is difficult for me to confirm positive correlation  between B and C expression. The positive side is that i could confirm the outcomes which support that B upregulates C
in sparse condition.
 i want to ask whether i can make a research story like this
: I got results which indicate that A acts as positive upstream molecule of B, which increases C expression so A regulates C under high cell confluency.
However, because transcription activity of B is very low under such dense cell density which makes demonstrating increased C expression by B very difficult, i induced enhanced transcription activity of B by seeding cells sparsely, and i could see that B really promotes C expression.  A seems to regulate C not only via B but also other factors in high cell confluecy.
For me my idea flow seems quite reasonable but i am concerned about my
quite subjective view. So I want to get some opinions from others.
Thank you for reading so long question!
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Hi agree with geetanjali. I feel that at high density cells are in position to form tight/adherent junctions which stabilizes other molecules and signaling cascade. If B activity is low under high density, I feel look at the phosphorylation and the type of phosphorylation involved under high and low density. At low density cells are apart with minimal cell connections therefore the nuclear localization of B indicates the property of B to regulate the proliferation and growth of cells. C is the binding partner for B and this interactions is possible in the absence of tight junctions. B is a co-transcription factor and is activated only when A is present. What I feel, wnt /beta-catenin, TGF/BMP-beta-catenin signaling will be useful to study and deriving insight.
Thanks
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We would like to be able to screen multiple signaling pathways at once with primary cells after stimulating with whatever stimulus we choose. Doing tons of western blots is not possible so hoping someone can provide an answer to what they have used to look at for example JAK/STAT, MAPK, PI3K, NFkB, etc all at once. So far I've seen R&D has a phosho-kinase array but do need some feedback for what has worked well for primary cells. Phosphorylation flow cytometry is also a possibility but would rather do a broad screen first to give us a clue of what pathways to study further. Thanks!
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We worked with anemic mice and required about 6 mice per group.  Luckily the arrays are very sensitive!
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Hi
Anybody was able to use siRNA to block STAT3 alpha specifically?
Thanx
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Hi Sarmad, I currently work for a company called siTOOLs Biotech (www.sitoolsbiotech.com) and we have a siPOOL (high complexity siRNA pool for reduced off-targets) for STAT3. We can look into designing a specific STAT3 alpha siPOOL for you. Please let me know if you'd be interested. You can reach me at catherine.goh@sitools.de Thank you
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Hello
while doing mitochondrial membrance potential assay b JC-1, what could be the best positive control?
Thanks
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As our colleagues, I suggest to you use the cccp (Carbonyl cyanide m-chlorophenyl hydrazone). CCCP causes an uncoupling of the proton gradient that is established during the normal activity of electron carriers in the electron transport chain. More info in http://www.abcam.com/cccp-ab141229.html 
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MYH9(NM-IIA) is known for regulating cell migration. It can express both on cytoplasmic and membrane. If I can down-regulate MYH9 expression on cell membrane, will it effect its function?
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In fact, I have found a secreted protein to co-localize with MYH9 on the membrane(Detected by confocal) and reduce MYH9 expression on the membrane(Detected by WB), and it has some oncogenic functions and effects cancer related pathway.
Thanks.
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In my experiment using C3H10T1/2 cells I found phosphorylation of PKA substrates at band size of In-between 75-100 and 100-150 and also at 150 position. What might be those target proteins?
I used CST antibody 'Phospho-PKA Substrate (RRXS*/T*) (100G7E) Rabbit mAb #9624'
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Hi, It will be very hard to predict the identity of the bands as there are too many possibilities. In addition, the bands coming up with the PKA-substrate antibody is in no way conclusive proof that they are being phosphorylated by PKA. There are many kinases which can phosphorylate at PKA-like motifs. I am not sure about your objective, but to verify that these are indeed PKA targets in your cell system, you may use some inhibitors (or activators like Forskolin, if the system permits). The predicted targets and known substrates can be found at -
If you really want to identify the phospho-proteins, you may have to some way fractionate the lysates (eg. size, substrate-motif antibody IP), combined with inhibitor controls and go for MS analysis after phospho-peptide enrichment.
Good Luck with your analysis.
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I would like model the synergistic effect of two ligands (one with high affinity and the second with low) onto the same receptor. Both are activators of the receptor. Do anyone have experience with that?
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You want to know when they bind to same allosteric site or orthosteric site?
Best,
AMB
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I am looking to test various cdk inhibitors in cell lines and would like to be able to measure relative effects by following levels of phosphorylated target proteins. For example, the retinoblastoma protein phosphorylated at T821 is often used as an in vitro measure of CDK2 activity. Could anyone help me with a analogous target for CDK1- preferably one that is specific for CDK1 activity (ie not typically phosphorylated by other kinases). Many thanks for any help  
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Do you have any reference(s) or your own observation showing that TNF-alpha or TGF beta support proliferation? Theoritically, genetic defects leading to genesis of malignancy may affect mechinery of apoptosis, most cancer cells, however,  are still sensitive to stimuli of death receptor induced apoptosis.  I tarted to work on TNF induced apoptosis almost twenty years ago.  When HeLa cells were challenged with TNF-alpha, bledding cells occurred at 1-2 h after the treatment.  TNF-alpha triggers two arms of reactions, apoptosis and induction of NFkappaB.  Apoptosis is induced only when de novo protein synthesis is blocked by cycloheximide. 
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I want to proof that my protein promotes the activity of PCNA. The interaction of my protein with PCNA and the subunit p50 of polymerase delta is known. After knockdown of my protein the proliferation is decreased. Therefore, I want to show that the absence of my protein reduce the activity of PCNA. Have someone an idea for an assay?
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EGFR induced Y211 phosphorylation of PCNA.
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I have a mitochondrial uncoupler that inhibits viral infection. I want to show that this inhibition is due to a lack of ATP. However, the respiratory chain is blocked. I know that ATP in medium is cytotoxic and has difficulties to enter in cells.
In what form should I add ATP to the cells so that it comes in and can be consumed directly without going through the mitochondrial chain? (consumed directly by kinases)
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I observed that mitochondrial uncoupler drastically decreased intracellular ATP concentration and also inhibited the viral infection. I would like to be able to add ATP to the cells in the presence of the uncoupler to see if the infection is no longer inhibited.
My problem is how to add ATP to the cells to compensate this decrease?
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How can one study chronic activation of a receptor (G-protein) with a short half life at the cell surface/high turnover to reduce over-activation? Does increasing the dose of agonist with time improve receptor recycling?
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It might depend on the activation pathway you want to measure. Receptors can often continue to signal after internalisation (e.g. MAPKs, and even sometimes G proteins). 
If internalisation is stopping the signal, then you could try inhibiting internalisation (e.g. the dynamin inhibitor dynasore or  use < 18 C temperature).
It is all very receptor subtype dependent - some don't seem to desensitise and/or internalise at all. 
What specifically are you trying to achieve?
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I am doing anti diabetic activity via insulin receptor activation pathway.
I have acess to NIT-1, 3T3-L1 and C2C12 cell lines. what are the method could I use for the checking activity?
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Thanks, both of you.
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I want some literature regarding AKT phosphorylation for insulin receptor activation
How can we do it?
on Which cell lines we can perform AKT phosphorylation.?
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I am new to performing MAP kinase and other signalling pathways, please can anyone help me with the drug incubation time I should start with when performing my western blot analysis? I like to know what times are ideal to consider during phosphorylation.
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Usually signalling responses are very short term. So you should start with an experiment of 5 or 10 minutes. I stimulate my cells for 5 minutes and I see a very dramatic signalling response that goes down with longer time. And if I have to see a response of a drug (inhibitor), I usually pre-treat my cells for 30 min.  So basically, you can do a time-dependent curve starting at 5 minutes but you shouldn't have to go to anything beyond an hour.
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I want to know a gene strongly altered by 17 beta estradiol in human brain endothelial cells, so that i can take that gene as a control for the system.
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The one of the most accepted controls is progesterone receptor (PR). Oestrogen stimulated PR expression in majority of oestrogen sensitive tissues, including brain tissues. there is a review with oestrogen signalling in brain that might help you to define oestrogen-responsive genes in brain. see the attached files as references. Oxytocin receptor was suggested as oestrogen-regulated in brain astrocytes as well. you might need to verify the data by yourself using available novel methods and new antibodies before the large scale experiments. Majority of papers with oestrogen signalling are published with cancer cells, so I am not sure what you will find in normal brain cells, however, the PR is the most likely target that I would try first..
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Wnt pathway
Promoter assays
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Thanks a lot! 
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I know that the LY294002 inhibit the activation of AKT by supressing its upstream key molecule PI3K. But how about perifosine? It is an alkyl-phospholipid. Does it inhibit the activation of AKT partially by directly binding to AKT? I have got a molecule which could totally reverse the inhitition of phosphorylation of AKT by perifosine but not LY294002. I was wondering the underlying mechanism. 
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Perifosine does not directly affect phosphoinositide 3-kinase (PI3K), phosphoinositide-dependent kinase 1, or Akt activity at concentrations inhibiting Akt phosphorylation and membrane localization. Our results demonstrate that Akt is an important cellular target of perifosine action. In addition, these studies show that the membrane translocation of certain PH domain-containing molecules can be greatly perturbed by the alkylphospholipid class of drugs and imply further that the PI3K/Akt pathway contributes to regulation of p21(WAF1/CIP1) expression.
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I would like to treat my cells with TSA to test effect on the mobility of nuclear protein. As a readout of TSA treatment, what should I check to confirm TSA worked?
Thank you
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Thanks a lot, Nianli and Eugen. I will try one of these antibodies.
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Like silenced c-met and ron will lead to inhibition of cell migration and invasion by p42/44mapk phosphorylation and reduce stat 3 activation in cancer cell line. With this pathway, can I apply it in diabetic cell line if I spot there is reduced activation of stat 3 and mapk then link it to less expression of ron?
Can I use it to explain the reduction of wound closure on cell proliferation and cell migration based on an assumption basis or as an indicator of what can that pathway leads to? Or even, what can the receptor do relate to cell migration?
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I am only interested on the tyrosine kinase receptors and their interactions in cell migration/ cell proliferation. So I wished to use proven mechanism to understand what is the role of that certain tyrosine kinase receptor can do in a rough manner on normal/diabetic cell line. 
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I am confused by the role of cAMP on adipogenesis and lipolysis. I know that when cAMP is high, it activates PKA and thus phosphorylates HSL and perilipin A to facilitate lipolysis, and when cAMP, insulin, and glucocorticoid present together, the "cocktail" can induce adipogenesis. But if both procedure require high cAMP concentration, how does the cell decide which way to go? It does not make sense that the cell differentiates and breakdown lipid simultaneously. Also, insulin is supposed to inhibit cAMP production through PI3K pathway, how come that these two show together in the body to stimulate adipocyte differentiation?
If there are any figures that help illustrate this problem, it is more than welcomed. 
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Dear Xiao,
I think reading the below manuscript about Regulation of Lipolysis in Adipocytes could help to take off your confusion.
Regards
Ehab El-Haroun, PhD
doi: 10.1146/annurev.nutr.27.061406.093734
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Hi all,
Through which pathway AKT is related to mTORC1 and 2?
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MTorc1 gets activated, indirectly, by AKT. On the other hand, mtorc2 partially activates AKT (phosphorylation of S473 of AKT). 
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i have a jmy protein and akt phosphorylates it but at what sequence site
i wish to compare the diffrent akt phosphyrlation site of jmy on diffrent species
please dont just provide a link give me some depth also as i am stupid. 
can you please provide me with the human jmy akt region to which akt phosphorylates then i can do the rest
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search litterature for consensus sites.
check possible site on your protein, they are usually verified by mutagenesis, mutatinc the T or S which is the target by A for instance should abolish phosphorylation
and may differ is different specy. Alignment using blast or clustal or other should help you.
to find your protein sequence with all annotations start with ncbi.nih.org or pubmed
go to gene first as there will be most info, then click on the protein number.
you can also look in uniprot database from there a lot of links will provide a lot of valuable info. Just type name in google you will have a starting point.
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JMY protein is phosphorylated by AKT i know AKT1 and 2 are anti apoptotic and AKT1 decreases cell motility whereas AKT2 increases motility but what does this mean for when these phosphorylate  JMY how do they effect apoptosis, cell motility and autophagy e.g.
i know phosphrylation of a protein affects its function but what does JMY phosphorylaion by akt1 or akt2 do in this context?
any help is appreciated, as i am not very bright could you explain your answer please in some depth 
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The protein JMY is known to function as p53 co-factor in regulating p53 functions. Therefore, it is conceivable that phosphorylation of JMY by AKT kinase impairs the ability of JMY protein to potentiate the p53-mediated anti-tumor functions that you mentioned. However, other possible explanations are likely.
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Hi all,
Is there any evidence that asparaginase provoke eIF2 kinase, PERK activation in cell lines? Thank you in advance
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hi there could you please tell me to what sequence on JMY does AKT kinase bind too i have checked on pathwaynet and JMY is indeed phosphorylated by AKT but what exact region in the sequence does it bind too?
any help would be appreciated
could you explain your answer in laymans terms as im not very bright and provide the website to which you found this infomation . 
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I am working on mRNA  dopamine receptor to see the changes of gene expression of these receptor  in rat brain in 3 different area
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There is no clear relationship. Only 30–40% of the variance of protein
abundance is explained by mRNA abundance in mammalian cells. Translation of mRNA is a complex and tightly-controlled process secondary to transcription, and is regulated by initiation and elongation factors, availability of amino
acids, and small RNA molecules such as microRNA and short interfering RNA.
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I wanted to know about the positive affects of using a natural polymer in terms of how it affects the Wnt pathway
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  • I have not examined this. But looking at the structure of polymer and its wound healing properties, and itsrole in mitochondrial biogenesis, one needs to examine its role in gkycosylation of proteins that promote transcriptionalactivators.. This field needs further examination. I am sorry, i am not more useful in this
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Do we have some webbased tool to do this? Or we just download each pathway gene list to find this? Because common signaling pathway could initiated by different receptors, maybe they want say something similar, does it possible for us to find tools to find this common targets? 
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That is what I want! Thank you Joseph.
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I'm looking for an inhibitor of Pi(5)P kinase, publications didn't help me a lot, maybe plumbagin but i'm not sure. I found many inhibitors for Pi3K and Pi4K but not for Pi5K and I need it to inhibit kinases and see effects on bacterial replication in eukaryotic cells. Thank you if you know what molecule can be used. 
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I guess, you can check these Target Report Cards in ChEMBL to find compounds with suitable activity:
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What are the cell lines I can use it for the experimental studies related to Aquaporin-2 and Vasopressin receptor, which is found in renal cells of the human?
I have seen in Wikipedia that  HEK 293 cells should not be used as an in vitro model of typical kidney cells.
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Thanks Emily... :) 
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Is there such a case that certain antibodies have limited access to the intra-cellular domain of its receptor and this results in a negative staining?
I have been struggling trying to detect signals associated with the intra-cellular β-subunits of the insulin-like growth factor receptor (IGF-1R). Using exactly the same protocol however, I got very good results showing a positive staining of extracellular α-subunits of IGF-1R. 
I have tried all the commercially available antibodies against IGF-1R beta-subunit which are applicable for immunohistochemistry, so I was wondering why this is the case.
Also, if my IHC results showed positive staining of α-subunits, can I conclude that the whole receptor is present, rather than saying only the α-subunits of the receptor are present?
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Dear Yinan,
As the target is a specific sequence present in the midst of the 1333 to 1366 AA at the c-terminus of IGF-IRβ subunit, localizing it could be a problem if the sequence is getting denaturated for some reason. 
1- whatever the cell-line you are using do not detach the cells by trypsinization. Keep the flasks on ice for 30 mins and then tap vigorously by hitting the flask to a wooden surafe gently. Use the physically detached ones for IHC/ICC.
2- . Insufficient cell permeabilization could be an issue as mentioned by Lars. 
3-  If these dont work, you can validate IGF-IRβ by tyrosine kinase inhibitors, I guess.
Will keep you posted on this in 24 hrs if i get something new.
Best,
AMB
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I want to induce blockade with DPCPX and SH58261 (Selective A1 and A2A adenosine receptor inhibitors, respectively) in cultured L6 rat skeletal muscle cells as a part of an inhibitory assay. Both inhibitors are insoluble in water and therefore, the solubility is given in DMSO (Dimethyl Sulfoxide). I just wanna know the best DMSO percentage (100% DMSO or a dilution such as 0.5% DMSO) efficient in preparing the inhibitory solutions in required concentration.
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Thank you Dr. Bruce, Samantha and Ernst for your valuable comments and recommendations. I hope to go ahead with a final concentration of 0.1% DMSO while using the same concentration with the vehicle (KRPH, pH 7.4), but without inhibitors as the control.
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Ezrin is a FERM domain cytoskeletal protein that in the inactive state assumes a conformation by which the N-terminal domain binds the c-terminal actin binding tail. I know phosphorylation leads to protein to open up and bind to actin filaments. How can I design a biosensor to detect activation state of Ezrin? what are the positive and negative control constructs?
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I agree with Elena on the control constructs and also that it may be challenging to make this approach work in Drosophila. I have not worked with Drosophila in this context, although FRET biosensors have been used previously in Drosophila so it may be possible (see for example http://www.sciencedirect.com/science/article/pii/S0898656807002069).  With regards to the controls; I am not sure of the consequences of constitutive/lack of phosphorylation of ezrin in Drosophila, but if there are severe effects on normal functioning such constructs may not be useful in vivo. If there is a way to pharmacologically block/stimulate activation it may be sufficient.
I would suggest optimizing and validating the biosensor in a heterologous expression system. Doing this would allow you to assess the feasibility of the approach without the challenges of imaging in tissue/intact animals.
Good luck with your study!
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Do you know if human α-Glucosidase have high homology with yeast α-Glucosidase ? thank you
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Thank you for your help Mikael and Francesco!
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The bands of WB suggesting the natural compound inhibited the phosphorylation of both Stat5 and Stat3. The extent of reduction of p-Stat3 and p-Stat5 is different, the level of p-Stat5 reduced more remarkable.
So, the questions is, can we just assume that the target of this compound is more likely to be Stat5 or the upstream of Stat5. Or in other words, this compound is more easily combine with Stat5 or the upstream protein of Stat5? If so, I want to know what methods we can use to further prove my assumption.
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When looking at STATs phosphorylation status is no longer a suffice readout for activity. The best way to prove an effect is my doing qPCR for target genes activated by stat5  and 3 , to show specificity towards STAT5. For upstream you can try probing for phospho  and total levels of the IL2 receptor /Jak pathway.
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I am trying to do a protein kinase c inhibition experiment in HeLa cells. I have tried 1uM and 500nM concentrations. 
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Hi Gregor,
Thank you for replying. I am targeting all of them. Basically looking to inhibit as many isoforms I can. Thats why I chose these pan inhibitors. I am using these inhibitors in context of chlamydia infected HeLa cells. One side effect that I see is induction of apoptosis but chlamydia is known to inhibit apoptosis in cell anyway. Hope this gives you a background of my work.
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Meaning, is there a loss of PTEN and/or an increase in upstream signaling to PI3K/Akt that would lead to above average phospho-Akt levels, as compared to other cell lines? Thanks y'all!
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Thank you Tomas!
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It has been well known fact that many receptor that activated upon ligand binding,but there are also reports that receptor internalisation happens ,
these two events specially,how they are regulated 
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Question was in general ,but specifically in B cell where BCR has two function 
1.internalization upon binding antigen :helpful in surface representation of antigen
2.signalling by BCR :HELPFUL IN PLASMA CELL DIFFERENTIATION
Confusing part is both part 1 and 2 are necesary for immune system to work,
Now question remains how this choice is made go by 1 or 2 or both ,
@ Cornelius Krasel
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I want to check role of Wnt signaling during differentiation of cardiosphere derived cells to cardiomyocytes. Wnt acts in time-dependent manner during differentiation. So, I want to add Wnt inhibitor  at different time points of differentiation induction.  Is IC50 value to be considered ? or have to check the expression of markers at different time periods of differentiation with different concentrations of inhibitor.
Kindly provide with proper protocol to determine the concentration
Thank you
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The basic biological concepts in life process including cellular differentiation is very simple on molecular accepts but not in signaling levels. Deriving the value in units and determining the concentration of inhibitor is surely vary from the age of your cells and obviously the quality. My understandings if not wrong, please start with more than 12X  and check it. Importantly keep remember the medium disturbances too, because there are many bond breaking events and joining too.
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In my view the concentration of the ligand is transmitted as the frequency of collision on the receptor. The higher is the concentration of the ligand the number of ligands hitting the receptor per second is higher. But more importantly the receptor should transmit the frequency of ligand collision as a frequency signal onto the next element of the signal transduction pathway, too. This is not as evident as we might think. The collision of the ligand impacts receptor structure, which should be repaired before the next collision. If this does not happen then the receptor will not be aware of the next ligand collision and will be unable to transmit the correct frequency and thus the correct concentration of the ligand. I note, that the information of the concentration is not transmitted, independently whether the receptor is still in its active state (that is continuously activates further elements of the signal transduction pathway) or it is blocked (and does not activate further processes) when the next collision arrives. Do you agree with this visualisation of receptor function? Please give your opinion
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I don't think it is true, bcoz collision frequency is independent of the nature of the ligand and receptors.  What is believe is that the lied ligand and receptor has particular binding affinity and site. so when the ligand binds with the receptor or analytes , tho HOMO -LUMO gap of the ligand changes which in turn changes the photophysical properties of the ligand. That is the basis of Ligand analyte interaction. With the increase in receptor concentration more number of ligands gets complex thus more increase in photophysical output. After all the ligands gets complexed there is no mre increase or decrease in signal attaining saturation point.
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Specifically ROR gamma 
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Dear Kolla,
I suggest first you establish a dose-response relationship with a known agonist for reference. Then you measure the dose-response with your compounds alone as well as together with the agonist. If a compound is an antagonist, you will see no change of basal activity if measured alone, while observe a parallel right-shift of the agonist dose-response curve when measured together. In case of an inverse agonist you also observe  a shift when measured together, but must observe a decline in the dose-response curve with increasing concentration below the basal level. This can be observed only if a measurable basal activity is present. If a potential inverse agonist compound is identified this way, then you might design binding assays to see where the compound binds and how it affects the binding of agonist (to differentiate the functional effect from an allosteric negative co-operativity effect), but this is a more complicated issue and you need purified protein for that.
Regards,
Karoly
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Hi All,
I have a bit of headache to find good downstream target gene to track for NFkB and Myc pathway activity.
What I did was treating A549 cell with inhibitors of NFkB (BAY 11-7082) and Myc (10058-F4) for 24 hours and then tracking the downstream gene expression through first generating cDNA (qScript cDNA SuperMix) and real time PCR (Fast SYBR Green Master Mix).
I have tried following list of known NFkB and Myc downstream genes but they show no differences (compare to GAPDH control) or even induction by the inhibitors even though the amplification looks good.
Myc: CEBPB, TERT, CCND2, PTEN, PCNA, TP53, RPL19, E2F1, EIF4A1, EIF4B, EIF4E
NFkB: AGT, CFB, CSF2, CSF3, IFNA1, IFNB1, TNF, LTA, IL6, IL8, ICAM1
I don't know what I did wrong so I get this weird result!
Any suggestion of good downstream targets and possible way for me to fix the experiment would be most appreciated!
Best,
Lyra
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please provide me any study related to the activation of phosphorylation of estrogen receptor alpha at serine 167 have any direct or indirect relationship with induction of apoptosis
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I came across  3  quite substantial papers on PubMed central:
PLoS One. 2012;7(6):e37736. doi: 10.1371/journal.pone.0037736. Epub 2012 Jun 29.
Polyphenols sensitization potentiates susceptibility of MCF-7 and MDA MB-231
cells to Centchroman.
Singh N(1), Zaidi D, Shyam H, Sharma R, Balapure AK.
Polyphenols as "sensitizers" together with cytotoxic drugs as "inducers"
cooperate to trigger apoptosis in various cancer cells. Hence, their combination
having similar mode of mechanism may be a novel approach to enhance the efficacy
of inducers. Additionally, this will also enable to achieve the physiological
concentrations facilitating significant increase in the activity at
concentrations which the compound can individually provide. Here we propose that
polyphenols (Resveratrol (RES) and Curcumin (CUR)) pre-treatment may sensitize
MCF-7/MDA MB-231 (Human Breast Cancer Cells, HBCCs) to Centchroman (CC,
antineoplastic agent). 6 h pre-treated cells with 10 µM RES/CUR and 100 µM RES/30 µM CUR doses, followed by 10 µM CC for 18 h were investigated for Ser-167 ER-phosphorylation, cell cycle arrest, redox homeostasis, stress activated
protein kinase (SAPKs: JNK and p38 MAPK) pathways and downstream apoptosis effectors. Low dose RES/CUR enhances the CC action through ROS mediated JNK/p38 as well as mitochondrial pathway in MCF-7 cells. However, RES/CUR sensitization enhanced apoptosis in p53 mutant MDA MB-231 cells without/with involvement of ROS mediated JNK/p38 adjunct to Caspase-9. Contrarily, through high dose sensitization in CC treated cells, the parameters remained unaltered as in
polyphenols alone. We conclude that differential sensitization of HBCCs with low
dose polyphenol augments apoptotic efficacy of CC. This may offer a novel
approach to achieve enhanced action of CC with concomitant reduction of side
effects enabling improved management of hormone-dependent breast cancer.
J Mol Med (Berl). 2011 Feb;89(2):181-91. doi: 10.1007/s00109-010-0698-y. Epub
2010 Nov 23.
Mammalian MST2 kinase and human Salvador activate and reduce estrogen receptor
alpha in the absence of ligand.
Park Y(1), Park J, Lee Y, Lim W, Oh BC, Shin C, Kim W, Lee Y.
Mammalian MST2 kinase plays an important role in cell proliferation, survival,
and apoptosis. In search of interacting proteins of MST2, we found that estrogen
receptor α (ERα) co-immunoprecipitates with MST2 and its adaptor protein human
Salvador (hSAV). Using reporter assays, we observed that overexpression of MST2
and hSAV leads to ligand-independent activation of ERα in human breast cancer
MCF-7 cells, which was attenuated by the knockdown of hSAV. Furthermore, using
truncated mutants of hSAV, we observed that the C terminus of hSAV is necessary
and sufficient for the induction of ERα transactivation. The expression of hSAV
and MST2 results in the phosphorylation of ERα at serine residues 118 and 167 and represses ERα expression. We then investigated the incidence of MST2 and ERα expression with other tumor biomarkers using commercially available tissue
microarrays. Among 40 breast cancer samples analyzed, 60% (24 out of 40)
expressed MST2. Nineteen among the 40 cases were MST2-positive and ERα-negative, implying a correlation between expressions of MST2 with loss of ERα in breast tumor samples. This study suggests that MST and hSAV act as novel co-regulators of ERα and may play an important role in breast cancer pathogenesis.
Int J Cancer. 2004 Mar 20;109(2):167-73.
Resveratrol modulates the phosphoinositide 3-kinase pathway through an estrogen
receptor alpha-dependent mechanism: relevance in cell proliferation.
Pozo-Guisado E(1), Lorenzo-Benayas MJ, Fernández-Salguero PM.
Resveratrol (RES), a natural phytoalexin, has antiproliferative activity in
human-derived cancer cells and in rodent models of tumor development. We have
previously shown that RES induced apoptotic death in estrogen-responsive MCF-7
human breast cancer cells. Recent data have indicated that the estrogen
receptor-alpha (ERalpha), through interaction with p85, regulates
phosphoinositide 3-kinase (PI3K) activity, revealing a physiologic, nonnuclear
function of the ERalpha potentially relevant in cell proliferation and apoptosis.
In our study, using MCF-7, we have analyzed the ability of RES to modulate the
ERalpha-dependent PI3K pathway. Immunoprecipitation and kinase activity assays
showed that RES increased the ERalpha-associated PI3K activity with a maximum
stimulatory effect at concentrations close to 10 microM; concentrations >50
microM decreased PI3K activity. Stimulation of PI3K activity by RES was
ERalpha-dependent since it could be blocked by the antiestrogen ICI 182,780. RES
did not affect p85 protein expression but induced the proteasome-dependent
degradation of the ERalpha. Nevertheless, the amount of PI3K immunoprecipitated
by the ERalpha remained unchanged in presence of RES, indicating that ERalpha
availability was not limiting PI3K activity. Phosphoprotein kinase B (pPKB/AKT)
followed the pattern of PI3K activity, whereas RES did not affect total PKB/AKT
expression. PKB/AKT downstream target glycogen synthase kinase 3 (GSK3) also
showed a phosphorylation pattern that followed PI3K activity. We propose a
mechanism through which RES could inhibit survival and proliferation of
estrogen-responsive cells by interfering with an ERalpha-associated PI3K pathway,
following a process that could be independent of the nuclear functions of the
ERalpha.
This article (below) has been retracted!!!
IKKepsilon phosphorylation of estrogen receptor alpha Ser-167 and contribution to tamoxifen resistance in breast cancer.
Guo JP, Shu SK, Esposito NN, Coppola D, Koomen JM, Cheng JQ.
J Biol Chem. 2010 Feb 5;285(6):3676-84
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I am trying to visualize calcium release in primary cells in culture over long periods of time.  basically we give a treatment at time 0 and want to do time lapse imaging of calcium release from the ER and its accumulation in the cytosol over a 24 hour period for example. 
Does anyone have any suggestions of how to accomplish this.  we tried using fura and oregon-green bapta-am, but they seem to be good only for acute calcium release. 
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Could you use different cultures and label each time you want to measure with the sensors you use?
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Hey there,
I found out that HEK293 cells are not useful for examining PI3-kinase mediated signaling, as they typically display constitutively active Akt, but no reference is cited. Does anybody know where can I find a reference for that source?
Thanks in advance!
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I agree with Divaker Choubey. Transformed cells often have major cell survival signaling pathways such as RAS/MAPK and/or PI3K/AKT.
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I've been using TOPFlash luciferase assays to measure Wnt signalling in HEK293T cells, in order to study the effect of transfecting a protein of interest (a transcription factor) on Wnt signalling. The assay uses a TOPFlash luciferase reporter, which has binding sites for Wnt transcription factor TCF and is expressed when the Wnt pathway is active, and a second (control) reporter, Renilla luciferase, which is expressed constitutively and is used to normalise data to correct for transfection efficiency and sample handling. The issue I'm having is, when I co-transfect my protein of interest alongside the two reporters, I get an increase not only in the TOPFlash reporter signal but also in the Renilla control reporter signal. Others have reported seeing a modulation of Renilla expression by experimental factors (first link below), which it is suggested could be due to factors interacting with regulatory elements in the TK (or other) promoter, and it was suggested that using a promoterless Renilla (pRL-Null) could overcome this problem (1st/2nd link). However, I have tried using the promoterless Renilla plasmid and found I still have the same problem. 
So my questions are these:
1) Why might my Renilla signal be increasing when I transfect my protein of interest (NB. Total protein levels in the cell, measured by BCA assay, do not appear to be increased in concert with the increase in reporter signal)
and, related to this
2) How is transcription initiated when there is no promoter? I can't seem to find anything on this, but transcription must be happening, because I'm seeing a substantial amount of signal with the Renilla construct.
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The completion of genome sequences from human and mice suggested a regulatory role of space (Chorobiology) in controlling biological phenomena and activities.  Consequently fundamental constants of mathematics such as π, the ratio of a circle's circumference to its diameter, should have a role in describing properties and establishing control in biology.
The Greek mathematician Archimedes who found π, developed a rigorous approach to approximating π. Archimedes observed that polygons drawn inside and outside a circle would have perimeters somewhat close to the circumference of the circle.
While π is an infinite number (never ending No), it relates to a symmetric geometrical image, the circle, but intriguingly Archimedes used polygons to measure the circumference of the circle.
What could be the meaning or the role of π in the control of signal transduction? And why π is an infinite number?
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pi (π), in mathematics, the ratio of the circumference of a circle to its diameter. The symbol for pi is π. The ratio is the same for all circles and is approximately 3.1416. It is of great importance in mathematics not only in the measurement of the circle but also in more advanced mathematics in connection with such topics as continued fractions, logarithms of imaginary numbers, and periodic functions. Throughout the ages progressively more accurate values have been found for π; an early value was the Greek approximation 31/7, found by considering the circle as the limit of a series of regular polygons with an increasing number of sides inscribed in the circle. About the mid-19th cent. its value was figured to 707 decimal places and by the mid-20th cent. an electronic computer had calculated it to 100,000 digits. Although it has now been calculated to some 2.6 trillion digits, the exact value of π cannot be computed. It was shown by the German mathematician Johann Lambert in 1770 that π is irrational and by Ferdinand Lindemann in 1882 that π is transcendental; i.e., cannot be the root of any algebraic equation with rational coefficients. The important connection between π and e, the base of natural logarithms, was found by Leonhard Euler in the famous formula e i π = - 1, where i = - 1art/square-root-of-negative-1.gif the square root of negative 1.
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Hi Everyone I am looking for antibodies for ER pathways that works in INS-1 cells or any other cells. There are lots of paper out there but I want to know if anybody have tried these antibodies in INS-1 cells and that has worked perfectly. The antibodies I am interested are as follows:
spliced-XBP1. p-PERK, PERK, p-IRE1 alpha, Total-IRE1 alpha, ATF4, ATF6 active.
Any information will be highly appreciated. Thanks in advance.
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UPR/ER stress is activated through an orchestrated interplay of signaling molecules from all three arms of the UPR pathway i.e. IRE1, ATF6, and PERK. This is why, it is critical to analyze signaling players in the context of the mentioned fact. Novus' ER Stress / UPR Antibody Pack contains sample size vials of the top markers namely: GRP78/HSPA5, pSer724 IRE1 alpha, total IRE1 alpha, XBP1, ATF6, and CHOP/GADD153. For scientific technical answers to over 15 similar questions, I would suggest reviewing our UPR and ER Stress FAQs at https://www.novusbio.com/support/upr-and-er-stress-faqs
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I want to test antioxidant drug in spontaneous colorectal cancer mouse model. By knocking down NLRP3 inflammasome, mice which already having spontaneous colitis  develops tumor in proximal colon at age 12, without giving any carcinogen. I am wondering whether my antioxidant will work on this model or not? I want to inhibit the ROS level in order to ameliorate the progression of colitis to cancer. 
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Hi Sonia,
To figure down your scientifically important problem of using a proper antioxidant, it is crucial to understand in your model peculiarities of a known “triangle problem” (relationships between NLRP3, macrophage ROS and tumor growth). Yes, in other models, the NLRP3 inflammasome, a protein complex controlling maturation of important pro-inflammatory cytokines interleukin (IL)-1β and IL-18, is also involved in tumorigenesis and metastasis of various cancers. However, a common denominator among the multiple damage-inducing agents that ultimately lead to activation of NLRP3 has not yet been identified. Recent study showed that ROS inhibitor such as DPI abolished IL-1β release in mouse macrophages by inhibiting NLRP3 gene expression (Bauernfeind F, et al. “Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome”. J. Immunol. 187:613–617, 2011). It was demonstrated that production of ROS has been suggested to act as a common event upstream of the NLRP3 inflammasome machinery. Because de novo translation of NLRP3 is an essential step in the activation of NLRP3, the role of substances that inhibit either ROS production or its oxidative activity was studied. Although NLRP3 inflammasome activation is unique among other known inflammasomes in its sensitivity to ROS inhibition was observed, it was found that this phenomenon is attributable to the fact that NLRP3 strictly requires priming by a proinflammatory signal, a step that is blocked by ROS inhibitors. Although these data do not exclude a general role for ROS production in the process of NLRP3-triggered inflammation and subsequent malignization, they would put ROS upstream of NLRP3 induction, but not activation.
Best regards,
Ilya
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Hello,
I am unable to find some nice data for my lecture on the role of lipids in cell signalling. Kindly help me if anybody has some nice, sufficient data/notes on this topic.
Regards
Dr. Arush
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Kindly go through
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I am doing an apoptosis analysis by flow cytometry. The cells were stained by AnnexinV and DAPI. The DAPI negative cells show an decreased FSC and increased SSC, suggesting an apoptotic phenotype. But the cells do not show increased Annexing V compared to the control cells. Anybody encountered that before? What is the possible explanation for that? This was not an reagent issue because the DAPI positive population were AnnexinV high, so the AnnexinV reagent was working.
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Hi,
Itseems like Annexin V is working but when you say you have not found any difference in Annexin V compared to the control cells that indicates either your treatment (Drug or any additional factor) to induce apoptosis has not worked or has no effect on Apoptosis.
There can be necrosis also occuring in the cells not just apoptosis. To rule out if necrosis is taking place, you can repeat the same experiment but with Propidium Iodide staining and Annexin V.
 Annexin +/ PI- = early apoptotic,     Annexin +/ PI+ = Late apoptotic
Annexin-/PI+ = Necrosis, Ann -/PI - = Alive cells
All the best.
Shalini Suresh