Substitution of CDK9 serine 90 for alanine reduces CDK9 association with large P-TEFb complex. 293T cells were transfected with Flag-tagged WT CDK9, CDK9 S90A and CDK9 S90D and also co-transfected with Cyclin T1-expressing vectors. At 48 hrs posttransfection, the cells were lysed with low salt (large complex – LC) buffer followed by high salt buffer (small complex – SC). CDK9 expression was analyzed by Western blotting. (A) Representative immunoblots. (B) Quantification of CDK9 in large (LC) and small (SC) complexes conducted in two independent experiments. (C) Co-immunoprecipitation analysis. 293T cells were transfected with Flag-tagged WT CDK9 and CDK9 S90A and also co-transfected with Cyclin T1 expressing vectors. At 48 hrs post-transfection, the cells were lysed; CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies, resolved on 10% SDS PAGE and analyzed by immunoblotting with antibodies for Hexim1, CDK9 and cyclin T1. Lane 1, mock-transfected control.