I am working with cancer cells and assess the effect of different compounds on NFkB pathway and apoptosis. As I observed phosphorylation of p65 upon treatment and a rescue of apoptosis by adding 2 different NFkB inhibitors, I assumed an unexpected involvement of NFkB as inducer of apoptosis. Not in line with this, I see a decreased activation of NFkB activitiy by using a commercially available NFkB reporter assay kit (Promega). Does anyone of you have a possible explanation for this? I would apprecciate any recommodations.
Thank you.
you may look to NF-kappaB driven genes (VCAM, CXCL8, IL-6 or others) by RT-PCR to have an idea about the real activity of NK-kB in your experimental system. NF-kB p-Ser phosphorylation is supposed to be an earlier event with respect to transcriptional activity, the different response in the two assays you found may be matter of timing. Regarding p56 phosphorylation, you should be sure that the total NF-kB levels are the same in the different samples (hou have to normalize with unphosphorylated NF-kB) To make things more clear (or more confused?) you may look also to IKB phosphorylation (at early time), total IKB protein levels, and NF-kB nuclear translocation. Good luck Raffaele
I think phosphorylation at different sites may result in varied targeting of the protein in a cell. In case of p65, it is nucleus bound protein after its release from cytotoxic complex with Ikb. On phosphorylation targeting might switch towards other vesicles degradation pathways. I recommend to test if MG132, inhibitor of protein degradation pathway rescues the effect of you observed. Thanks
Dear all,
thanks for you suggestions - hope to reveal clearly the contribution of NFkB in my tumor cells. Am still confused, but countless reviews underline the fact, that NFkB has different faces under different circumstances regarding cell signaling and different stimuli. Will now also check phospho-IkBa and acetylated Ly310.
Thanks and I am still open for further suggestions.
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