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Fig. 6. Cdk5 induces p53 stabilization by phosphorylation. (A) GST-p53 or GST-p53-SDM (a mutant p53 with alanine substituted for Ser15, Ser33 and Ser46) proteins were incubated with active Cdk5-p35 recombinant protein in a kinase reaction buffer, with roscovitine added to inhibit Cdk5 activity. Levels of p53 phosphorylation and Cdk5 kinase activity were examined by autoradiography, and the amount of GST-p53 was assessed by Coomassie Blue staining. (B) H1299 cells were transfected with the indicated plasmids, as described in Fig. 3D,E. At 36 hours post-transfection 20 µM MG132 was added and cultures were incubated for an additional 8 hours. (i) Total p53 was immunoprecipitated with a polyclonal antibody and detected with a monoclonal antibody. The ubiquitylated form of p53 is indicated. EGFP was used as the transfection control. (ii) To detect the association of p53 with Hdm2, cell lysates were immunoprecipitated with anti-p53 and then immunoblotted with anti-Hdm2, with 5% of the cell lysates used as input controls. (C) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, and then treated with 5 µM mitomycin C or an equal volume of vehicle for 8 hours in the presence of 20 µM MG132. (i) Total p53 was immunoprecipitated with a polyclonal antibody and detected with a monoclonal antibody. The ubiquitylated form of p53 is indicated, and 5% of the cell lysates was used as input controls. (ii) For detecting the association of p53 with Hdm2, cell lysates were immunoprecipitated with anti-p53 and then immunoblotted with anti-Hdm2, with 5% of cell lysates used as input controls.
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