Fig. 4.

The ATR kinase is required for Ser-15 phosphorylation after PUVA treatment. A, diploid human fibroblasts were pretreated with different concentrations of wortmannin for 30 min followed by treatment with HMT (1 μg/ml) and UVA (30 kJ/m²). Cells were incubated for 6 h in the presence of wortmannin before the cells were harvested, and Ser-15 phosphorylation of p53 was analyzed by Western blot. β-Actin was used as loading control. B, diploid AT fibroblasts were treated with different concentrations of HMT followed by UVA light (30 kJ/m²). Cells were harvested 6 h later, and the levels of total p53 protein and Ser-15 phosphorylated p53 were determined by Western blotting. C, human diploid fibroblasts were microinjected into the nucleus with either IgG or anti-ATR antibodies. After 1-h incubation, the cells were treated with HMT (1 μg/ml) and UVA (30 kJ/m²), and 2 h later, the cells were fixed and stained for Ser-15 phosphorylation of p53. Arrows indicate microinjected cells. D, quantification of the percentage of cells staining positive for phosphorylated Ser-15 of p53 treated as in C. The total numbers of injected cells were 100 and 102 for IgG and anti-ATR, respectively. The error bars represent the S.D. of results from at least 5 different experiments with approximately 20 microinjected cells for each.