Figure 10
From: Myc stimulates cell cycle progression through the activation of Cdk1 and phosphorylation of p27
Conditional knock out of Cdk1 totally abolishes cyclin B1-dependent phosphorylation of p27. (a) Protein extracts from Cdk1 conditional knock out MEFs and controls treated with 0.6 μM 4HT or vehicle (DMSO) for different periods of time. Expression of cyclin A2, cyclin B1, Cdk1 and Cdk2 are shown. Actin levels were measured as loading control. (b) Cdk1lox/lox MEFs transduced with a MYC-IRES-GFP construct or the corresponding empty vector. Myc overexpression assayed by western blot along with cyclin B1 and cyclin A2 levels. (c) In vitro kinase assay of cyclin B1 immunocomplexes on His-p27 in Cdk1lox/lox and control Cdk1+/lox treated with 4HT for 72 hours using p27 as substrate. (d) In vitro kinase assay of immunoprecipitated cyclin A2 from Cdk1lox/lox MEFs transduced with a Myc-IRES-GFP construct or the corresponding empty vector and treated with 4HT for 72 hours. (e) Expression of Myc and Cdk1 in Cdk1lox/lox MEFs transduced with a Myc-IRES-GFP construct and treated with 0.6 μM 4HT or DMSO for 72 hours. Actin levels were determined as loading control. Signal densitometry quantification of the kinase assays are shown below each lane. Kinase buffer with His-p27 was used as negative control (No IP) and unspecific IgG was used as negative control for the specificity of the antibody used for immunoprecipitation.
