We cannot use luciferase reporter gene in our experimental settings and we would like to quantify p65 activation by a mean that enable a standardization by the number of viable cells. We thought about qPCR which will be really quantitative but we are still wondering of a specific target activated by p65. Would you recommend IkB?
I would suggest Nrf2 as p65 unidirectionally antagonized its transcriptional activity.
May be both IκB and Nrf2.
Good luck
I would suggest pro-inflammatory genes that are target of p65 like iNOS or TNF-alpha by qPCR methods at relatively short times (3-6hrs).
Alternatively you could try with specific phosphorylation of p65 that is directly correlated to p65 activation (prior to nuclear translocation). We used Phospho-NF-kB p65 (Ser536) antibody (3033, Cell Signaling) by Western Blotting and normalized against total NF-kB (no phosphorylated-p65) as well as with a loading control (e.g. beta-actin).
Best regards
Leonardo
Can I ask why you cannot use luciferase? Might be worth looking into something like species-specific NIS (sodium iodide symporter) for use with SPECT/PET imaging. It would be noninvasive and nonimmunogenic and give you clear 3D biodistribution of your gene expression (viable cells only).
Hope that helps!
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