Ser175 mediates the binding of Tat to CDK9 and BRD4.

Abstract

<p>(A) Induction of Tat expression in latently infected Jurkat 2D10 cells. WCEs were prepared from 2D10 cells and treated for the indicated times with PMA (50 ng/ml), TNF-α (10 ng/ml), or a combination of anti-CD3 (0.125 µg/ml) and anti-CD28 (1 µg/ml) mAbs. The extracts were then subjected to Western blotting using a Tat monoclonal antibody.(B) Relative binding of Tat and BRD4 to P-TEFb. Top: Western blots of WCEs (left panels) or FLAG-CDK9 immunoprecipitates (right panels). Top two panels show an experiment detecting BRD4 association with FLAG-CDK9 while the bottom three panels show a separate experiment detecting CycT1 and Tat association with FLAG-CDK9. Graph shows the relative levels of co-precipitated BRD4, Tat, and CycT1 normalized to corresponding total CDK9 levels. Data are from three different experiments. Error bars: ± standard error of the mean. (C) Tat-dependent and signal-dependent dissociation of P-TEFb from 7SK snRNP. 293T cells stably expressing FLAG-tagged CDK9 were transiently transfected with HA-tagged Tat. Immunoprecipitation was performed using anti-FLAG antibody followed by immunoblotting using antibodies against CycT1, CDK9, HEXIM1, LARP7, and Tat.</p