Figure 5

CDK2-directed siRNA blocks Tat phosphorylation. A, HeLa cells were infected with Adeno-Tat (lanes 2 and 3). At 4 hours post infection, cells were transfected with siRNAs targeting CDK2 (lane 3) or non-targeting control pool (lane 2). Lane 1 – control cells. At 48 hours post-infection cells were labeled with (³²P)-orthophosphate for 2 hours. Whole cell extract was subjected to immunoprecipitation with anti-Flag antibodies, resolved by 15% Tris-Tricine SDS-PAGE, and transferred to polyvinylidene fluoride membrane. A, immunoblot of the membrane with anti-Tat monoclonal antibodies using the 3,3'-diaminobenzidine enhancer system. B, autoradiography of the membrane on Phosphor Imager screen. C, quantification of the panel B. Position of Tat is indicated by arrow. The position of light chain of IgG recognized in anti-Flag immunoprecipitates by anti-mouse HRP-conjugated secondary antibodies is indicated by asterisk.