FIG. 11.

Effect of PC3 on the protein kinase activities of cyclins-CDKs in vivo. (A) Characterization by Western blotting of NIH 3T3 cultures infected with retrovirus carrying the PC3 coding region or with empty vector (from supernatants of BOSC23 cells transfected with the pBABE puro-PC3 or pBABE puro vector, respectively). Equal amounts of proteins were loaded. (B) In vivo activity of CDK2 and CDK4 in NIH 3T3 cells infected with the PC3 retrovirus or with the empty retrovirus, as indicated. Equal amounts of proteins, from NIH 3T3 lysates of cultures infected with PC3 retrovirus or empty retrovirus, were immunoprecipitated with normal rabbit serum (NRS) or with anti-CDK2 and anti-CDK4 antibodies and then assayed for GST-Rb phosphorylation in 50-μl reaction mixtures by measuring incorporation of ³²P. Samples were loaded in SDS-PAGE gels and transferred by electrophoresis to a nitrocellulose filter. This was analyzed for the presence of phosphorylated GST-Rb by a PhosphorImager (upper panels). The immunoprecipitated samples were checked for the presence of equal amounts of CDK2 and CDK4 proteins by Western blot analysis of the nitrocellulose filter (lower panels). INF., infected; I.P., immunoprecipitation.