Figure 4

CDK2-directed siRNA inhibits CDK2 expression. A, CDK2-directed siRNA inhibits expression of CDK2. HeLa cells were transfected with siRNAs targeting CDK2 (lane 3) or non-targeting control pool (control, lane 2). Lane 1, untransfected cells. At 48 hours post-transfection cells were lysed and cellular extracts were resolved on 12% Tris-Tricine SDS-PAGE and analyzed by immunoblotting analysis with antibodies against CDK2, CDK9 or α-tubulin. B, quantification of the CDK2 expression in panel A using α-tubulin expression level for normalization. C, CDK2-directed siRNA inhibits enzymatic activity of CDK2. CDK2 was precipitated from cellular extracts prepared from HeLa cells transfected with siRNAs targeting CDK2 (lane 2) or non-targeting control (lanes 1 and 3). Lanes 1 and 2, precipitation with rabbit anti-CDK2 antibodies. Lane 3, precipitation with rabbit preimmune serum. Immunoprecipitates were incubated with γ-(³²P)ATP and recombinant Tat (see Methods), resolved on 12% Tris-Tricine SDS-PAGE and analyzed by autoradiography on Phosphor Imager. Position of Tat is indicated.