Reduction of X-IR–induced phosphorylation of p53 in L-4.

(A) PCR products of exon 64 (338 bp) from L-4, LCL-wt1, and pcDNA3-YZ5/8921T plasmid were digested with AlwNI. The 249-bp and 89-bp fragments are expected in the 8921C>T mutation, but not in the wild-type sequence. (B-C) Cells were harvested at various minute intervals after 5-Gy X-IR and analyzed by Western blotting with anti-p53–Ser15 phosphospecific antibody. α-Tubulin was used as a control for protein loading. Levels of phosphorylated p53–Ser15 as detected by Western blotting were quantified by scanning densitometry and were corrected for α-tubulin content, and standardized to those of LCL-wt1 (= 1) (B) and LCL-wt2 (C). Data were expressed as the mean (± SE) of 2 independent experiments (bottom graph in C). In panel C, the histogram is shown only for the data at 30 minutes because the data at 60 minutes were less informative due to the activity of ATR.