I'm working on cancer biology in AMPK pathway.

I use some AMPK activator like metformin, and when I treat that activator, I detect the phospho-AMPK (Thr172) is increasing in western blot data.

And I treat the Compound C in same sample, but the phospho-AMPK is extremely upregulated!

I saw same result in many times. I don't know how to troubleshoot.

I never saw in any paper that using Compound C. All of the references show phospho-AMPK is decreased when Compound C treat.

I already try pretreatment method, and ATP adding method.

Is there anything that can I do??

If someone use the Compound C, please tell me detail procedure..