PP2A dephosphorylates CDK9 in vitro. A, Dephosphorylation of CDK9 by PP2A and PP1. Recombinant CDK9/cyclin T1 was incubated with γ-(32P) ATP to allow autophosphorylation (lane 1). The kinase activity was blocked by 7 mM EDTA and CDK9 was used as a substrate for PP1 (lanes 2 and 3) or PP2A (lanes 4 and 5). Dephosphorylated CDK9 was resolved on 10% SDS-PAGE and quantified on PhosphoImager (lower panel). B, Phosphorylase-a phosphatase activity of PP1 and PP2A at concentrations corresponding to panel A, presented as the amount of phosphorylase-a remained in the reaction after the treatment with the phosphatase. C, Pre-treatment with PP2A increases autophosphorylation of CDK9. Recombinant CDK9/cyclin T1 was incubated without (lane 1) or with PP1 (lanes 2 and 3) or PP2A (lanes 4 and 5) at concentrations corresponding to Panel A. After incubation, the phosphatases were blocked with 1 μM okadaic acid and CDK9/cyclin T1 was subjected to the autophosphorylation with γ-(32P) ATP (lanes 1 to 5). Phosphorylated CDK9 was resolved on 10% SDS-PAGE and exposed to the PhosphoImager screen.