Figure 5
From: Halogenated imidazole derivatives block RNA polymerase II elongation along mitogen inducible genes
Differential inhibitory effects of DMAT and TBBz on CDK9 activity in vitro. (A) CDK9/Cyclin T1 proteins pulled down from nuclear extracts (anti-CDK9+anti-cyclin T1, Santa Cruz Biotechnology, D-7 and H-245, respectively) were used in CDK9 autophosphorylation reactions without or with DMAT or TBBz. Autophosphorylated proteins were resolved by SDS-PAGE, transferred to PVDF membrane and immunostained by anti-CDK9 antibody (upper panels) and scanned using a Phosphorimager (lower panels). Similar patterns were obtained in 3 different experiments. (B) Recombinant fusion full-length human CDK9+CyclinK proteins, co-expressed by baculovirus in Sf9 insect cells (Abcam, ab70320) were used in autophosphorylation assays as above. (C) Complexes of CDK9/Cyclin T1 proteins pulled down from nuclear extracts or (D) human CDK9+CyclinK protein were used in kinase assays, with or without DMAT or TBBz. The heptapeptide YSPTSPS was used as a substrate. The reaction mixtures were applied to acidic hydrolysis of γ32P- ATP followed by phosphomolybdate extraction, and 32P-phosphopeptide was determined by liquid scintillation spectrophotometry. Results are expressed as a percentage of kinase inhibition and represent means ± S.D. of 2 separate experiments.