Figure 4
From: Myc stimulates cell cycle progression through the activation of Cdk1 and phosphorylation of p27
Cdk1 mediated phosphorylation of p27 at the Thr-187. (a) Left panel: In vitro kinase assay of Cdk1 immunoprecipitated from Cdk2−/− Lv141 and Cdk2−/− Myc MEFs using His-p27 as substrate. Phosphorylation levels of p27 at the Thr-187 were detected using a phospho-specific antibody. Right panel: Levels of the immunoprecipitated Cdk1 assayed in the experiment of the left panel measured by western blot. (b) Total lysates from Cdk2−/− Lv141 and Cdk2−/− Myc MEFs transfected with empty vector (Ev) HA-Cdk1-wt (WT), HA-Cdk1-DN (DN) or non-transfected (NT). Total Cdk1 and exogenous Cdk1 are shown (anti-Cdk1 and anti-HA antibodies respectively). Actin levels were used as loading control. (c) Total Cdk1 immunoprecipitated from protein extracts showed in the left panel and immunoprecipitated exogenous Cdk1 levels measured with anti-HA. (d) Kinase activity of Cdk1 wild-type (WT) and Cdk1DN (DN) on Thr-187 of p27. Immunocomplexes were treated with 10 µM purvalanol A (Purv A) or vehicle (DMSO) in the kinase assays when indicated. Signal densitometry quantification of the kinase assays are shown below each lane. Unspecific IgGm was used as negative control for the specificity of the antibody used for immunoprecipitation.
