Figure 3

Sam68 overexpression decreases G1 Cdk activities and Rb phosphorylation. A. Cells were synchronized in mitosis by nocodazole treatment or in G0 by serum deprivation and released into G1 by drug removal or serum replacement, respectively. At the indicated times after release cells were lysed in 3T3 lysis buffer and immunoblotted with antibodies against Sam68, cyclin D1, cyclin E, Cdk2 or phospho-Rb. B. Lysates from cells released from mitosis for the indicated times were subjected to immunoprecipitation with anti-cyclin E or -Cdk4 antibodies. Immunoprecipitates were assayed for in vitro kinase activity (KA) with histone H1 or GST-Rb as substrates, respectively. Indicated below the Cdk4 assay gel are relative ³²P incorporations into Rb per lane, as determined by phosphorimager analysis. Whole cell lysates (WCL) or immunoprecipitates (IP) were also Western blotted (WB) with anti-Cdk2 antibodies. Results are representative of three separate experiments.