FIG. 5.

CDK8 is required for T3-dependent activation of DioI mRNA expression. (A) RNAi knockdown of Mediator subunits in α-2 cells. Whole-cell extract was prepared from α-2 cells transfected with siRNAs specific for MED1, MED17, CDK8, cyclin C, or a nonspecific scrambled control siRNA and then probed with the specific antibodies indicated on the right. The immunoblots were then stripped and reprobed with antibodies against α-tubulin. (B and C) Loss of CDK8 or cyclin C inhibits T3-dependent gene expression. α-2 cells transfected with either control or Mediator-specific siRNAs were treated with or without T3 (10⁻⁷ M) for 1 h (B). Total RNA was then extracted processed for quantitative RT-PCR in real time using primers specific for DioI. Alternatively, α-2 cells were transfected with 2×TRE-tk-Luc along with either control or Mediator-specific siRNAs for 48 h and then treated with or without T3 (10⁻⁷ M) for 16 h (C). The cells were then harvested and assayed for luciferase activity, which was normalized against expression from a cotransfected β-galactosidase control vector. In the lower panels, equal amounts of cellular lysate from the harvested cells were probed by immunoblotting using the antibodies indicated on the right.