S6K in vitro kinase assay

S6K1 proteins purified from transfected cells were used for in vitro kinase assays. Two micrograms of bacterially purified His-S6 was used as the substrate. The reactions were performed in the presence of 1 mM adenosine triphosphate (ATP) and kinase reaction buffer [10 μM tris-HCl (pH 7.5), 10 mM MgCl₂, 0.1 mM EDTA, and 2 mM dithiothreitol] at 30°C for 30 min, and the reactions were stopped with 3× SDS loading buffer and then subjected to immunoblot analysis.