Fig. 4.

Soluble mutant MED12-containing Mediator and reconstituted kinase modules show no defect in CDK8 incorporation. (A) Nuclear extracts from control CNTL, FG, or Lujan patient cells were subjected to IP with goat IgG (IgG*), rabbit IgG, or antibodies specific for CDK8 (goat) or MED30 (rabbit) as indicated. Immunoprecipitates were processed by WB analysis using antibodies specific for the indicated Mediator subunits. Input corresponds to 20% of the nuclear extracts used for IP. (B and C) Lysates from High Five insect cells coexpressing the indicated combinations of epitope-tagged kinase module subunits CBP-MED13, MED12-HA (WT/R961W/N1007S), CDK8-FLAG, and 6HIS-CyclinC were subjected to IP with FLAG-specific antibodies. Note that because CDK8 expression is limiting relative to other kinase module subunits during viral infection, WT and mutant MED12 derivatives are thus present in excess levels at the input concentrations used in IPs. (B) Immunoprecipitates were processed by WB analysis using the indicated antibodies. (C) Immunoprecipitates were incubated with a purified GST-3xCTD substrate (corresponding to three heptapeptide repeats from the RNA polymerase II large subunit) in the presence of [γ-³²P]ATP before resolution by SDS/PAGE and visualization of ³²P-labeled GST-3xCTD by phosphorimager analysis (Upper) and total input GST-3xCTD by Coomassie blue staining (Lower). The amount of ³²P-labeled GST-3xCTD catalyzed by each kinase module derivative was quantified and expressed relative to the amount catalyzed by WT MED12-containing module (100%). Values represent the average ± SEM of three independent experiments.