Discuss the network in signaling pathways, trying to collaborate and interchange ideas.

• Zemin Wang

  Methods for AhR, CAR, PPARa activity measurement in mice liver

  Does anyone know good techniques/kits available for measuring CAR, AhR, PPAR-a activity in mouse liver homogenates? Any suggestions, links, papers or protocols are heartily appreciated.

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    Zemin Wang replied

    Thanks for your information. We had originally thought to look at whether these NRs are activated after treatment with their agnoists by looking at the activity of their major target protein. ButThanks for your information. We had originally thought to look at whether these NRs are activated after treatment with their agnoists by looking at the activity of their major target protein. But what your talked might be a good alternative for us to go. Thanks again!

    3 days ago
• Rocío Ortiz-Butron

  It is posible to measure ROS and lipoperoxidation in adipose tissue?

  We want to know if there are some alterations in the adipose tissue related with oxidative stress in women than have been having problems getting pregnant.

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    Rocío Ortiz-Butron replied

    The samples will take from visceral adipose tissue from woman. This will be realize by a medical doctor. We will put the samples in frozen ice (-70ªC), until we can measure the ROS orThe samples will take from visceral adipose tissue from woman. This will be realize by a medical doctor. We will put the samples in frozen ice (-70ªC), until we can measure the ROS or lipoperoxidation techniques.

    6 days ago
• Hanaka Mimura

  What is the most famous gene whose transcript levels are regulated by ERK?

  I want to know whether or not the gene of my interest is sensitive to PD98059 (MEK/ERK inhibitor) and I have this distinct feeling that it is not. Even if the gene was indeed insensitive to PD98059,I want to know whether or not the gene of my interest is sensitive to PD98059 (MEK/ERK inhibitor) and I have this distinct feeling that it is not. Even if the gene was indeed insensitive to PD98059, I still have to check other genes regulated by ERK to see if my PD98059 was really working. I know the most credible way to do this is to do the Western blot analysis of phospho-ERK, but the easiest way is RT-PCR and I heard c-Fos is regulated by ERK. Can someone please tell me other genes regulated by ERK?

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    Gabriela Gorelik replied

    Those are different things. To check if your inhibitor is working, you need to measure p-ERK by WB. And regarding the genes that are regulated by ERK, there are many. ERK activates AP1 transcriptorThose are different things. To check if your inhibitor is working, you need to measure p-ERK by WB. And regarding the genes that are regulated by ERK, there are many. ERK activates AP1 transcriptor factor that regulates different genes, as i.e. Dnmt. So, you need to do RT-PCR to measure the expression of your specific gene after the incubation with the inhibitor.

    18 days ago
• Ana Virgen

  I'm looking for a technique to assess protein ubiquitination in a nematode

  How do you assess the state of ubiquitination of a protein?

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    Piers D Nash replied

    These biochemical approaches may be difficult in nematodes unless you are looking broadly at ubiquitination in the whole organism and can do biochemistry on extracts. Genetic approaches to studyingThese biochemical approaches may be difficult in nematodes unless you are looking broadly at ubiquitination in the whole organism and can do biochemistry on extracts. Genetic approaches to studying post-translational modifications tend to be a bit limited. There are systems for studying UPS activity in C. elegans using GFP fusion constructs (e.g.. doi:10.1038/emboj.2011.195). You can find a summary of the worm Ub system with LOF information and links for RNAi reagents at http://www.wormbook.org/chapters/www_ubiquitinpathways/ubiquitinpathways.html

    20 days ago
• Shishir Gupta

  Could anyone give me some suggestions on how signalling networks can be converted into boolean models?

  Cell signalling.

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    Gabriel Weinreb replied

    Depends on what exactly you mean. Do you have a model network written in terms of differential equations, for instance? Or you want to build a network from scratch? There are a lot of publicationsDepends on what exactly you mean. Do you have a model network written in terms of differential equations, for instance? Or you want to build a network from scratch? There are a lot of publications about this so you need to be more specific. How big your network is? If it is about tens to hundred elements, Boolean approach probably would be too coarse grained. The approach usually effective when you have big number of elements.

    Apr 13, 2012
• Shakti Singh

  In lymphocytes, Ras can be activated by multiple pathways. Why is there the necessity of multiple pathways in Ras activation?

  In lymphocytes, Ras signalling is known for the transmission of various receptor mediated signal to the cell to proliferate, differentiate, negative selection, induction of cytokine production etc.In lymphocytes, Ras signalling is known for the transmission of various receptor mediated signal to the cell to proliferate, differentiate, negative selection, induction of cytokine production etc. There are multiple pathways of activation of Ras signalling. Why do the lymphocytes not just use only a single mechanism to activate the Ras signalling instead of so many different pathways of activation?

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• Tanmoy Ray

  Does passage number affect signalling pathways in cell lines?

  I am working with two different cell lines. The first one (3T3-fibroblasts) is a genetically engineered one. It is stable line that over-expresses our protein of interest. The other one is HL-1I am working with two different cell lines. The first one (3T3-fibroblasts) is a genetically engineered one. It is stable line that over-expresses our protein of interest. The other one is HL-1 cardiomyocytes, and they express the protein normally. Currently both our cell lines are very old now, in their P~70s; and I am having some problems to reproduce some results that I used to get when the cells were in their P~20/30s. So, I am wandering if the regulatory signalling pathways have been altered. Any advice/comments would be much appreciated.

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    Renata Jasinska replied

    As a principle: Propage your cell line first and prepare multivials stock about 1mln cells/each in LN2 from the first passage. Store in at least two different LN2 tanks. Keep processed material fromAs a principle: Propage your cell line first and prepare multivials stock about 1mln cells/each in LN2 from the first passage. Store in at least two different LN2 tanks. Keep processed material from the first passage as your internal control for further experiments.

    Mar 21, 2012
• Nishi Chandra

  System biology approaches towards modelling the inhibition of a DNA binding protein signaling pathway in cancer cells.

  I want to ask about how to model it. Is there a proper method for modelling this pathway? Is there a proper software to model it and if yes how to do it? I have collected all the interactions ofI want to ask about how to model it. Is there a proper method for modelling this pathway? Is there a proper software to model it and if yes how to do it? I have collected all the interactions of genes in this pathway, I just need a proper guideline to start my modelling. Could you help me out?
• Lucas D Tilley

  Ideas On How To Identify Kinase Substrates

  I would like to start a discussion on the different approaches out there for the identification of kinase substrates. All general ideas on the topic, totally theoretical thoughts on ways to identifyI would like to start a discussion on the different approaches out there for the identification of kinase substrates. All general ideas on the topic, totally theoretical thoughts on ways to identify kinase substrates, established methods with demonstrated success, or computational or wet bench approaches are welcome. Thanks!

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    Jonas Cicenas replied

    Y2h is absolutely useless...it's an artificial crap even for the interactions, why then going so far and spending money to find out if these crappy "targets" are phosphorylated. No self respectingY2h is absolutely useless...it's an artificial crap even for the interactions, why then going so far and spending money to find out if these crappy "targets" are phosphorylated. No self respecting database ever considers y2h as solid evidence....

    Mar 9, 2012
• Maria-Rosa Sarrias

  PI3K activity

  We would like to determine PI3K activity in monocytes. Could anybody help us with the methodology? Or, alternatively - is there any available kit that works fine? Thank you

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    Carlos Guillen replied

    Hi. I determined the PI3 kinase activity by a radioactive method. It was published in Endocrinology (Guillen C et al. Endocrinology 2006). I hope that i can contribute to your work. However, thereHi. I determined the PI3 kinase activity by a radioactive method. It was published in Endocrinology (Guillen C et al. Endocrinology 2006). I hope that i can contribute to your work. However, there are some methods not using radioactivity. So, as it has been suggested by other people you could use it. In contrast, the sensitivity could be lower that that with radioactivity. Good luck!!. Carlos

    Mar 7, 2012
• Jonathan M. Shillingford, Ph.D.

  A database of cellular signaling pathways

  http://stke.sciencemag.org/cm/

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    Beatriz Alves Guerra replied

    Tanks!!!

    Jan 24, 2012
• Akhilesh Shukla

  Variability of adenyl cyclase

  Why protein kinase show variability in Heart muscles (contraction) and in lung (tracheal relaxation/bronchial dilation ) while in both places adenyl cyclase is stimulated.
• Hiba Shabir

  is there any evidence of signaling through ROS in A1AR mediated aortic contraction in pathological conditions?

  A1ARs have been found to produce cellular responses through a number of effector systems such as inhibition of adenylate cyclase, enhanced glucose uptake, increased ionistol phosphates etc. InA1ARs have been found to produce cellular responses through a number of effector systems such as inhibition of adenylate cyclase, enhanced glucose uptake, increased ionistol phosphates etc. In general, these responses have been found to be mediated by pertussis toxin sensitive G-proteins. However, the first known effector of A1AR was inhibition of adenylate cyclase. Experiments conducted on mice have shown that the activation of A1ARs causes contraction through PLC in the A1AR wild-type. The A1AR coupled to Gi/o protein is known to regulate signaling pathways in various tissues, including the modulation of PLC activity, inhibition of PLA2 and adenylate cyclase, activation of K+ channels, and inhibition of Ca2+ channel.In general, the activation of A1AR mediates its effect via the following signaling pathway: A1AR →PLC-βIII →PKC-α→ p42/p44 MAPK phosphorylation →contraction
• Md Shadab

  Subversion of host cell signaling by leishmania, an intracellular protozoan parasite.

  Leishmaniasis is is a vector (sand fly) born parasitic disease. Leishmania is a protozoan parasite which resides in macrophage ones get entry into its host . I would be happy if any one can join meLeishmaniasis is is a vector (sand fly) born parasitic disease. Leishmania is a protozoan parasite which resides in macrophage ones get entry into its host . I would be happy if any one can join me in discussing how this parasite avert the macrophages effector function and persists in it till dissemination to other cell. Manipulation of signaling machinary, responsible for microbicidal and effector function, by this parasite would be the focus of this discussion.

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    Md Shadab replied

    Can any body explain that although the parasite L . donovani evades activation of the three MAPKs (P38, ERK1/2, JNK1/2) in naive BMM , it has shown production of IL-10 in naive macrophage fromCan any body explain that although the parasite L . donovani evades activation of the three MAPKs (P38, ERK1/2, JNK1/2) in naive BMM , it has shown production of IL-10 in naive macrophage from peritoneum. The work is done in vitro. Second query is wheather the intact parasite do really interact with the TLRs present on macrophage's surface as siRNA against some TLRS has reported that the response as earlier observed was not the same when the particular TLRs were knocked down.

    Jan 13, 2012