Figure 5From: The F-box protein SKP2 mediates androgen control of p27 stability in LNCaP human prostate cancer cellsEffect 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.Back to article page