Figure 5
Functional characterization of Cyclin T1 mutant proteins expressed in human cells. (A) Coimmunoprecipitation of Cdk9 and Hexim1 with Gal4-CycT1 proteins. Mutants in red are reproducibly deficient for Hexim1 binding. Gal4-CycT1 mutant proteins were transiently expressed in HEK293 cells. Immunoprecipitation was performed with anti-Gal4 antibodies. Immunoprecipitated proteins were detected on Western blot with Cdk9, Cyclin T1 and Hexim1 antibodies. (B) CTD kinase assay associated with immunoprecipitated Gal4-CycT1 proteins. (Left) Incorporation of 32PO4 in a CTD4 peptide increases with incubation time (min.). (Right) Western blots showing that immunoprecipitates used for kinase assays contained the same quantities of Cdk9 and Gal4-CycT1 proteins. (C) In vivo assays of P-TEFb activity. Expression of a luciferase reporter gene driven by 5 Gal4 UAS upstream a minimal HIV promoter is activated by cotransfected Gal4-CycT1 proteins in HEK 293T cells. Fold transactivations are relative to luciferase activity in cells that do not express Gal4-Cyclin T1 proteins. Data result from three independent experiments with standard error bars. Western blots show proteins detected in lysates of typical experiments using an anti Cyclin T1 antibody. Gal4-Cyclin T1 migrates above the endogenous Cyclin T1.