Figure 3.

UF-linked mutations in MED12 disrupt its CycC–CDK19 binding and stimulatory activities. A and B, lysates from insect cells co-expressing baculovirus-produced CycCH6, MED12–HA (WT or mutant as indicated), and either CDK8-FLAG (A) or CDK19–FLAG (B) were subjected to IP with FLAG epitope-specific antibodies. FLAG-specific IPs were processed by Western blot (WB) using the indicated antibodies (top panels) or subjected to in vitro kinase assay prior to resolution by SDS-PAGE and PhosphorImager analyses (bottom panels). Input corresponds to 10% of cell lysate used in IP reactions. Horizontal arrows indicate the presence of specified Mediator subunits in parallel reactions. WB 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–MED12 WT 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–MED12 WT 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).