Effect of Rap on the cell growth (A), cdk2 kinase activity, p70 S6 kinase activity, cyclin E- and A-dependent kinase activity (B), cdk4 kinase activity, cdk6 kinase activity. cyclin D2- and D3-dependent kinase activity (C) in exponentially growing ED-40515(−) cells. (A) ED-40515(−) cells were incubated with or without 10 nmol/L Rap for 28 hours, then fixed with 70% ethanol for 1 hour and stained with 50 μmol/L acridine orange in PBS/0.1% glucose for 1 hour. The relative DNA content was determined by a flow cytometric analysis. (B) ED-40515(−) cells (4 × 106 cells) cultured with or without 10 nmol/L Rap for the indicated times were harvested and immunoprecipitated with anti-cdk2 antibody, anti-p70 S6 kinase antibody, anti-cyclin E antibody, or anti-cyclin A antibody. The kinase activity of the immunoprecipitates was assayed with histone H1 or S6 peptide as a substrate. Each column represents the average of triplicate samples with ± SD. (C) ED-40515(−) cells (4 × 106 cells) cultured with or without 10 nmol/L Rap for the indicated times were harvested and immunoprecipitated with anti-cdk4 antibody, anti-cdk6 antibody, anti-cyclin D2 antibody, or anti-cyclin D3 antibody. The kinase activity of the immunoprecipitates was assayed with GST-Rb as a substrate. Each column represents the average of triplicate samples ± SD.
