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Fig. 5. Cdk5 activity induces phosphorylation of p53 at residues Ser15, Ser33 and Ser46. (A) GST-p53 protein was incubated with active Cdk5-p35 recombinant protein in a kinase reaction buffer, and phosphorylation of specific residues was determined by western blot analysis using the indicated phosphorylation-specific antibodies. The level of GST-p53 was assessed by immunoblotting with anti-GST. (B) SH-SY5Y cells were treated with 5 µM mitomycin C or 2 mM SNP for the indicated times, and cell lysates were subjected to western blotting with the indicated antibodies. (C) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, and then treated with 5 µM mitomycin C or 2 mM SNP for 9 hours in the presence of 20 µM MG132. Total proteins were subjected to western blotting using the indicated antibodies. (D) SH-SY5Y cells were transiently transfected with mock vector or vectors encoding Cdk5-p35, Cdk5-p25 or Cdk5(D144N)-p25, and the amounts of the indicated proteins were determined by immunoblotting. (E) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, treated with 15 µM wortmannin for 1 hour, and then treated with 5 µM mitomycin C for 6 hours in the presence of 20 µM MG132. (i) The amounts of site-specific p53 phosphorylation were determined by immunoblotting. (ii) The results displayed in the bar graphs represent quantitatively analyzed data from three independent experiments, and are given as the mean ± s.d. For the quantitative analysis, each blot was normalized with respect to the level of total p53. *P<0.05. (F) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, treated with 15 µM wortmannin for 1 hour, and then treated with 5 µM mitomycin C for 6 hours. The amounts of p53 and GAPDH were determined by immunoblotting.
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