Dear All
I have used inhibitors of protein kinases A & G (H89 and KT5823) at concentrations of 1, 2, and 5 microM on a human cancer cell line. the aim was to investigate whether the effects of a certain drug treatment involves PKA or PKG. Results obtained so far showed that these inhibitors did not modulate the drug treatment-induced changes in protein expression levels or promoter activities. Although the frequently used concentration of either inhibitors in previously published reports is 1 microM, what would be a suitable positive control to validate these findings and concluding that neither PKA nor PKG is involved?
comments are welcome
thanks

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as your chosen inhibitors lack selectivity, it is absolutely necessary to demonstrate the effects on the molecular level; you may check specific transphosphorylation activity of PKA and PKG: design it for yourself (f.i. kemptide and radioactive ATP) or use a kit (ELISA, FRET-substrates etc),

or check specific cisphosphorylation activity with phospho-specific antibodies with IB

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what are the specific inhibitors do you recommend?

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I would also try KT5720 to inhibit PKA;

as these inhibitors were shown to have IC50 or Ki in the nanomolar range, they inhibit other kinases in the mikromolar range; to show the inhibition of PKA or PKG, it should be demonstrated by the aforementioned assays

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