FIGURE 3.

CDKN1C can inhibit CDK7 and CDK9 kinase activity toward the RNA pol II CTD. A, CDK9 was immunopurified from extracts of control H1299 cells or H1299 cells infected with Ad-p57 and subjected to kinase assays using bacterially produced GST-CTD as substrate. A single “+” sign indicates that 250 μg of cell extract was used for IP, whereas “++” indicates 500 μg was used. Following 30-min incubations, reactions were subjected to SDS-PAGE and Western blotting using the indicated antibodies. The Western blot signal using phospho-serine specific antibodies indicated the relative level of kinase activity toward CTD. B, CDK7 was immunoprecipitated from 30 μg of cell extract, and the kinase reaction was carried out for 20 min. C, H1299 cells were infected for 48 h with the indicated combination of viral vectors to express cdkn1c (p57) or CDK7. Overexpression of CDK7 rescues CTD-Ser-2 phosphorylation in the presence of excess cdkn1c. A similar viral vector for CDK9 was not available.