Signal-dependent phosphorylation of CDK9 at Ser175.

Abstract

<p>(A) Detection of Ser175 phosphorylation by Western blotting after 1 h PMA (50 ng/mL) stimulation for wild type CDK9 and the T186A and T186D mutants. FLAG-CDK9 carrying the wild type sequence, or the S175A, S175D, T186A, or T186D mutations was stably expressed in latently infected Jurkat 2D120 cells using the MSCV retroviral expression system. Top panel: Whole cell extracts used for immunoprecipitation were immunoblotted for total CDK9. Note the slower migration of the ectopically expressed FLAG-CDK9 compared to the endogenous CDK9. Bottom three panels: Anti-FLAG-CDK9 immunoprecipitates were screened by immunoblotting for CDK9, pThr186, and pSer175 using a polyclonal antibody derived using a 19-residue peptide carrying a pSer175 epitope. (B) Validation of the epitope specificity of the pSer175 CDK9 antibody by peptide blocking. Purified antibody was pre-incubated overnight with pSer175 peptide epitope prior to immunoblotting anti-FLAG-CDK9 immunoprecipitates derived from control Jurkat T-cells, or 2D10 cells expressing FLAG-CDK9 before and after stimulation for 1 hr by PMA.</p