PKA kinase assay with radiolabeled ATP

Control and PKAcat knock out HEK293 cells were treated with hydralazine for 2 h. Cells were lysed in lysis buffer (50 mM Hepes, pH 7.7, 1.5 mM MgCl₂, 0.2 mM Na₃VO₄, 1 mM EGTA, 150 mM NaCl, 10% Glycerol, 100 mM NaF, 50 mM beta Glycerophosphate, 0.1% NP40) containing a protease inhibitor cocktail via sonication, 2× for 10 s. Cell debris was removed by centrifugation (15,000 × g, 10 min) at 4 °C and supernatants were used for PKA kinase assays after protein measurement. Reactions were initiated by adding 20 microgram histone 2B (protein substrate), 50 micromolar ATP ([Ƴ−32P] ATP 10,000 cpm/pmol), 10 mM MgCl₂, 10 mM HEPES pH 8.0, 1 mM DTT and 1 mM benzamidine for 1 h at room temperature⁶⁴. The reaction was stopped by adding 5x Laemmli sample buffer followed by heating at 100 °C. Samples were loaded on 4–12% Crit XT Bis-Tris gels (Bio-Rad). Gel staining, drying and scintillation counting were performed⁶⁴. The histone band from the lane lacking cell extract was subtracted to correct for background.