Determination of CDK9 residues dephosphorylated in cultured cells. A, 293T cells were transfected with expression vectors Flag-tagged CDK9 (lanes 2 and 3), CDK9 T186A mutant (lanes 4 and 5) and CDK9 C8A mutant (lanes 6 and 7). Cells were labeled with (32P) orthophosphate in the absence (lanes 2, 4 and 6) and in the presence of 100 nM okadaic acid (lane 3, 5 and 7). Lane 1, mock transfected cells. Cellular extracts were immunoprecipitated with anti-Flag antibodies, resolved by 10% SDS-PAGE and transferred to PVDF membrane. Upper panel shows expression of CDK9 determined by probing the membrane with anti-CDK9 antibodies using 3,3'-Diaminobenzidine enhancer system. Lower panel is an autoradiogram of the membrane exposed to phosphor imager screen. B, quantification of the Phosphor Imager panel.