FIG. 8.
CDK8/cyclin C specifically phosphorylates the Pol II CTD in vitro. (A) Recombinant human CDK8 and cyclin C expressed in Sf9 cells via baculovirus were purified and stained with Coomassie blue. (B) Purified recombinant CDK8/cyclin C phosphorylates GST-Pol II-CTD in a concentration-dependent manner (see Materials and Methods for further information on the in vitro kinase assay). (C to E) H7 inhibits CDK8/cyclin C kinase activity. (C) GST-Pol II-CTD was preincubated with various concentrations of H7 prior to incubation with CDK8/cyclin C in the presence of [γ32]ATP. (D and E) GST-Pol II-CTD was incubated with CDK8/cyclin C together with unlabeled ATP in the presence or absence of H7 (10 μM). The reaction mixtures were resolved by SDS-PAGE and then probed by immunoblotting using antibodies specific for the Pol II CTD, phosphorylated Pol II CTD Ser-2P, or phosphorylated Pol II CTD Ser-5P.