Figure 3

Cell cycle analysis of cyclin D2 and p21/waf1. (A) ChIP assays were performed using G₀ cells (0 hour) and G₁/S cells (6 hour) as described in the methods section. Cyclin A primers, specific for cyclin A promoter positions -135 to -113 and +13 to +33, were used to amplify DNA obtained from IPs using antibodies for E2F4, p300, and p130. PCR products were run on a 1% agarose gel and visualized with EtBr. Lane 1 is molecular weight marker and lanes 3 and 8 are control IgG. (B) Cells were synchronized at G₀ by serum starvation for three days, followed by stimulation with complete media (containing 10% heat inactivated FCS) and collected at 0, 2, 4, 6, 8, and 10 hours. One hundred micrograms of total cellular protein from CEM, C81, human fibroblasts (HF), mouse embryonic fibroblasts (MEF), and NIH-3T3 cells were prepared, separated by reducing SDS-PAGE on a 4–20% gel, and blotted with anti-cyclin D2 rabbit polyclonal Ab. The antigen-antibody complex was detected as described in the methods section. (C) Cells were synchronized at G₀ and processed as described above, with the exception that anti-p21/waf1 rabbit polyclonal antibody was utilized for western blotting. (D) Cells were serum starved for 3 days, stimulated, and samples collected at appropriate time points. Kinase assays were performed using GST-Rb as described in the methods section. Representative results of three independent experiments are shown here. (E) Immunoprecipitations and control western blots for part D were performed as described above. NS depicts non-specific bands.