FIG. 2.

CDK8-Mediator facilitates TR-dependent transcription in vitro. (A and B) Immunodepletion of Mediator from HeLa cell nuclear extracts, as shown by results of immunoblot analysis of HeLa cell nuclear extracts incubated with either anti-MED1/anti-MED6 antibodies or preimmune serum (mock). Specific antibodies used for immunoblotting are indicated to the right of the panels. (C) Mediator depletion abrogates TR-dependent transcription. Transcription was measured in vitro by incubating the TRE₃Δ53 reporter gene in Mediator-depleted (ΔMED) or mock-depleted nuclear extract together with purified baculovirus-expressed TRα, RXRα, and T3 (10⁻⁷ M) as described previously (4). (D) Purified CDK8-Mediator restores TR-dependent transcription in a concentration-dependent manner. In vitro transcription assays were performed exactly as described for panel C, except that increasing concentrations of purified CDK8-Mediator were added to the reaction mixtures as indicated.