Signal Transduction - Science topic

Goodwin G. Jinesh Hi Goodwin. Can you elaborate what is an ARP2/3 activation assay?

Dr. Dror,

Yes, you will have to separate cytosol from the nucleus for p65 but not for ikkb.

Regards.

Athanasios Kesidis sir, I have a doubt,how can we experimentally prove if a particular gpcr is activated upon receiving a signal molecule? I know one method is to measure the level of intracellular secondary messengers..otherwise how can we prove that experimentally?

Hello Lee Yx

Fetal bovine serum is widely used as a universal and representative additive for the culture of countless types of primary and immortalized cells. The precise composition of FBS is unknown because this additive comprises various unidentified, naturally occurring small molecules and proteins which would also include EGF in fluctuating proportions. The utilization of FBS at a concentration of 10%, is currently regarded as a gold standard practice in cell culture protocols, but this concentration may not be optimal for inducing signaling pathways like the one you mentioned namely, EGF activated PLC induced PIP2 hydrolysis, continuously in cells cultured in medium that contain serum.

When cells are treated with EGF you need to starve them of serum because this will help to remove the knowns and the unknowns present in culture, reduce analytical interferences, and provide more reproducible experimental conditions. Moreover, it will reduce basal cellular activity and make the population of proliferating cells more homogenous, since they withdraw from the cell cycle to enter the quiescent G0/G1 phase. So, it is necessary to serum starve the cells before any type of treatment as it helps to induce cell cycle synchronization.

Best.

Saurabh Mandal thank you very much for your descriptive answer.

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

I'd suggest Biorender

@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.

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.

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.

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.

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

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.

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.

I will suggest WB STAT5 for better result

The mycoplasm contamination can be a problem in cell signaling pathways.

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.

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.

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.

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.

I believe the beta chain has a JAK site, so it can also induce an inter-cellular response.

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:

Thank you so much, Adil! It looks amazing!

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.

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:

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

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. 

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

We worked with anemic mice and required about 6 mice per group.  Luckily the arrays are very sensitive!

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

For positive control,  you should use CCCP (125 µL , 50 mM in DMSO) which reduces MMP. Please check the link below.

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.

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.

You want to know when they bind to same allosteric site or orthosteric site?

Best,

AMB

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. 

EGFR induced Y211 phosphorylation of PCNA.

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?

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?

Thanks, both of you.

You can check above mentioned papers!

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.

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..

Thanks a lot! 

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.

Thanks a lot, Nianli and Eugen. I will try one of these antibodies.

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. 

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

MTorc1 gets activated, indirectly, by AKT. On the other hand, mtorc2 partially activates AKT (phosphorylation of S473 of AKT). 

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.

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.

www.jbc.org/content/early/2017/02/.../jbc.M116.768408.full.pdf?..

Hi,

Check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1446077/

that might help!

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.

That is what I want! Thank you Joseph.

I guess, you can check these Target Report Cards in ChEMBL to find compounds with suitable activity:

Thanks Emily... :) 

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

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.

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!

Thank you for your help Mikael and Francesco!

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.

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.

Thank you Tomas!

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

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.

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.

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

Hello

Links here will help you

Good Luck 

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.

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

Could you use different cultures and label each time you want to measure with the sensors you use?

I agree with Divaker Choubey. Transformed cells often have major cell survival signaling pathways such as RAS/MAPK and/or PI3K/AKT.

I had a same issue. Look at those articles. 

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.

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