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is my lost protein is bcoz of cell death or my treatment worked??? - (Oct/07/2010 )

Hi everybody,
i am in a confusion how to interpret my result. i explain wat i did, my aim is to identify the signalling through which my gene is being made express (which is an overexpressed gene in cancer). to do this i treated the cancer cells with different inhibitors of signalling, in that i found the NFKb inhibitor has reduced my genes expression with respect to time, i mean i could see a gradual decline in the expression in course of time (on SDS PAGE),,,also this drug kills cell as well, my time points are from 30 mins to 6 hrs with 10 micromolar conc of the inhibitor. i even normalized with tubulin and PhosphoMAPK both seems to be unchanged and well quantified. so with this can i say the sharp decline in the expression of my gene of interest is drug induced or still it is bcoz of dying cells. shd i hav to do any other assay to confirm this result ??? ur suggestions will help me a lot. thanks in advance.



It seems rather unlikely that the decrease in expression you see is to be related with cell death, given that the two controls you have done are unchanged. But if you want to make sure that apoptosis is not the cause of the protein loss, you could blot for clived caspases or parp, which could monitor apoptosis.


thanks a lot madrius, i try to execute ur suggestion.