Fig. 4. Cdk5 activity mediates efficient nuclear accumulation of p53. (A) SH-SY5Y cells were pretreated with 10 µM Cdk2/5 inhibitor or an equal volume of vehicle for 30 minutes and then incubated with 5 µM mitomycin C for 6 hours. (i) Cells were double-labeled with Hoechst (blue) and anti-p53 (red) and then analyzed by fluorescence microscopy. (ii) Percentage of cells showing cytoplasmic distribution of p53. Data are the mean ± s.d. from three independent experiments. ^*P<0.0001. (B) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, and then incubated with 5 µM mitomycin C for 6 hours. (i) Nuclear and cytoplasmic fractions were prepared as described in the Materials and Methods, and subjected to immunoblotting with the indicated antibodies. Lamin B1 and -tubulin were used as nucleus- and cytoplasm-specific controls, respectively. (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 quantitative analysis, each blot was normalized with respect to the internal controls, lamin B1 and -tubulin. ^*P<0.05.