Currently, I've been working on a compound that is observed in papers to have inhibitory effect on Bcl-2 and Bax protein. However, we wanted to use the clinical concentration (higher concentration than in papers) and observed that in western blot assays, the Bcl-2 protein expression was decreased. Does this mean that not only does the compound inhibits the protein-protein interaction (as proven by co-IP assay), it could also inhibit the expression in a transcriptional level?
Is it possible that the high dose was toxic to the cells, resulting in less protein expression overall? Did you normalize the level of Bcl-2 with the total protein concentration or a housekeeping protein?
Hi J. Antes
I would agree with Adam. It could be that the lower levels of Bcl-2 is from an alternative effect of the drug. For instance we found that compounds that you believe hit Bcl-2 are infact inhibiting/activating p53 or ERK1/2 these ofcourse have effects on Bcl-2. You might want to check those too which may give you an idea of mediator effects of your compound on Bcl-2.
Blake Balcomb Adam B Shapiro thank you for your answers. Yes I did normalize the target proteins with Beta actin, and yes, we found out that the expression was decreased. We haven't checked the p53 and ERK1/2 Blake Balcomb but could be a possible route. Thank you for your suggestion. We shall consider it.
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