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
Hi Alexander, to test CDK1 activity you can use the MPM2 antibody that recognizes an abundant broad-spectrum phosphoepitopes on CDK1 substrates. I have used mouse anti-MPM2 (#05-368) monoclonal Ab (Millipore, Billerica, MA, USA) and it works very well! I hope my answer is useful for you. Good luck!
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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