Figure 2.

MED12 is an allosteric activator of CycC–CDK19. A and B, lysates from insect cells co-expressing baculovirus-produced MED12–HA, CycCH₆ (WT or mutant as indicated), and either CDK8–FLAG (A) or CDK19–FLAG (B) were subjected to IP with FLAG-specific antibodies. FLAG-specific IPs were processed by Western blot (WB) using the indicated antibodies (top panels) or incubated with [γ-³²P]ATP and purified GST–CTD prior to resolution by SDS-PAGE and PhosphorImager analyses (bottom panels). Input (IN) corresponds to 10% of cell lysate used in IP reactions. Horizontal arrows indicate the presence of specified Mediator subunits in parallel reactions. WBs were quantified and levels of MED12 and CycC in each IP were normalized to those of CDK8 (A) or CDK19 (B) and expressed relative to their corresponding normalized levels in the CDK8 (or CDK19)–CycC WT–MED12 IP (middle panel). ³²P-Labeled GST–CTD levels in each IP–kinase reaction were quantified and expressed relative to the level in the CDK8 (or CDK19)–CycC WT–MED12 IP. Data represent the average ± S.E. of 3 independent experiments. Asterisks denote statistically significant differences versus WT (Student's t test, ***, p < 0.001).