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Fig. 2. Cdk5 activity increases in the nucleus, and Cdk5 interacts with p53. (A) SH-SY5Y cells were treated with 5 µM mitomycin C for the indicated times, and nuclear fractions were subjected to immunoblotting. Cdk5 kinase activity was determined by autoradiography using histone H1 as a substrate. (B) SH-SY5Y cells were transfected with a vector encoding Flag-Cdk5 and then treated with 5 µM mitomycin C or vehicle for 6 hours. Cell lysates were immunoprecipitated with antibodies against p53 or control IgG. Membranes were immunoblotted with anti-Flag and/or anti-p53 antibodies, with 5% of the cell lysates used as input controls. (C) Lysates from SH-SY5Y cells treated with 5 µM mitomycin C for 3 hours were used for pulldown with GST-p53 or GST, and probed with antibodies against Cdk5 or GST. (D) H1299 cells were transfected with vectors encoding Flag-Cdk5, His-p35 or p53 as indicated. Cell lysates were immunoprecipitated with antibodies against Cdk5 or control IgG, and then immunoblotted with anti-p53, anti-His and/or anti-Flag, with 5% of the cell lysates used as input controls.
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