Effects of P-TEFb inhibitors on the kinase activity of P-TEFb in vitro and on the large form of P-TEFb in cells. In vitro P-TEFb kinase assays were performed using recombinant P-TEFb, Pol II CTD or DSIF in the presence of increasing concentrations of seliciclib (A) or flavopiridol (B). The kinase reactions were resolved by SDS-PAGE and the amount of incorporated γ-32P-ATP was quantitated with a Packard InstantImager. (C and D) Glycerol gradient analysis of HeLa37 cells treated with DRB. (C) HeLa37 cells were treated with increasing amounts of DRB for 1 hour and lysed to extract both forms of P-TEFb from the nucleus. The lysates were subjected to glycerol gradient sedimentation and the fractions were examined by immunoblotting for Cdk9 and cyclin T1. (D) The Cdk9 and cyclin T1 signals in the free (fractions 3–6) and large (fractions 8–11) forms of P-TEFb were calculated and plotted as a function of the concentration of DRB used in the cell treatment.