Effect of SKP2 overexpression on cell cycle progression and CDK2 kinase activity (A) LNCaP cells were maintained in the absence or presence of 10 ng/ml MIB for 72 h. MIB-treated samples were subsequently infected with Ad-SKP2 or Ad-SKP2-ΔF for the indicated periods, after which cells were processed for flow cytometry. Percentage of cells in G1, S, and G2/M phases was blotted in a diagram. Note that SKP2 overexpression is not sufficient to override MIB-induced G1 arrest. The experiment was independently performed three times. (B) LNCaP cells were arrested with 10 ng/ml MIB for 72 h followed by infection with Ad-SKP2 for the indicated periods (24–72 h). Cell lysates were prepared and expression of the indicated proteins was determined by immunoblotting. (C) CDK2-dependent histone 1 (H1) in vitro kinase activity and cyclin E/p27 interaction were assessed in the same lysates used in (B). CDK2 immunoprecipitates were incubated with H1 in the presence of 32P-ATP and reaction products were separated by gel electrophoresis and detected by autoradiography (left panel). Parallel cyclin E immunoprecipitates were examined by immunoblotting for the amount of precipitated cyclin E and co-precipitated p27 (right panel). H1 kinase activity and the amount of p27 coprecipitated with cyclin E were quantitated using Totallab software (graph below blots). The input amount of H1 is shown in the lower panel on the left.
Lu et al. BMC Cell Biology 2002 3:22 doi:10.1186/1471-2121-3-22