i want to study the expression of NF-kB and TGF-b and STAT3 in flow cytometry sorted MDSCs cells (case vs control) and in tumor tissue in order to determine their activation status in inflammatory/pathological conditions. is it good idea to study the expression by PCR. should i have to check their downstream proteins by western blot/ihc?
You can look at NF-kappa activity by multiple ways:
1) Total IκBα levels to look for degradation as a sign of IKK activation
2) Phosphorylation of IκBα; p IκBα S32/S36
3) Phosphorylayion of IKKs by WBing for IKKalpha/beta, or in vitro kinase assays as mentioned by Esra.
4) Phosphorylation and translocation of p65.
5) Gene reporter assays like NF-KB response element controlled luciferase vectors. I have also cloned and transduced cells with NF-KB induced GFP vectors and measured NF-kappa induction by flow cytometery with great success.
6) You can also do qPCR and look for IKK-activated genes like cFLIP, IL6, IL1beta, TNF, etc
If you are expecting a consistent baseline differences between your cells these methods can be used at any time point, but if you plan to induce IKK and then measure activity, make sure to perform a time-course study to identify the peak signal point in your cells of interest. Because of the oscillatory nature of the signaling pathways like IKK and JAK/STATs this is crucial and will save you a lot a hassle.
Good luck
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