Figure 1

Inhibition of CDK9 activity and phosphorylation by CDK2-directed siRNA. (A-B) Inhibition of CDK2 expression and CDK9 activity. Lysates from 293T cells transfected with CDK2-directed (lane 1) or control siRNA (lane 2) were analyzed by immunoblotting for the expression of CDK2, CDK9, cyclin T1 and tubulin (panel A)Panel B, the cells were also co-transfected with Flag-CDK9 and cyclin T1 expression vectors. Lysates were immunoblotted with antibodies against CDK2, Flag and α-tubulin as loading control (upper panel) or immunoprecipitated with anti-Flag antibodies (lanes 1 and 2) or non-specific IgGs (lane 3) (lower panel). CDK9 activity was analyzed with GST-CTD as substrate. Lane 4, control recombinant CDK9/cyclin T1. GST-CTD is shown as Coomassie stain. Quantification is shown as average from three independent experiments. (C) Inhibition of CDK9 phosphorylation. Lysates from 293T cells transfected with CDK2-directed (lane 3) or control siRNA (lanes 1 and 2) and also with a vector expressing Flag-CDK9 WT as in panel B then pulse-labeled with (³²P) were immunoprecipitated with anti-Flag antibodies (lanes 2 and 3) or with non-specific IgGs (lane 1), resolved on 10% SDS PAGE and exposed to a Phosphor imaging device (upper panel) or analyzed by immunoblotting with anti-CDK9 antibodies (lower panel). (D) No effect on CDK9 Thr186 phosphorylation. Lysates from 293T cells transfected CDK2-directed (lane 1) or control siRNA (lanes 2 and 3) and then co-transfected with vectors expressing Flag-CDK9 WT (lanes 1 and 2) or Flag-CDK9 T186A (lane 3) were immunoprecipitated with anti-Flag antibodies and analyzed by immunoblotting with Thr186 phospho-specific or anti-CDK9 antibodies.