Interaction between hRad9 and p53. (A) Endogenous association of hRad9 and p53. Immunoprecipitation and Western blot analysis were performed using lysates of 293 cells. Anti-c-kit antibody was used as a negative control. (B) Immunoprecipitation using cell lysates of 293 transfectants with FLAG-tagged wild-type or phosphorylation-defective RAD9 plasmids. Each cell lysate was immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-p53 (top) or anti-FLAG (bottom) antibody. HC, IgG heavy chain of the anti-FLAG antibody. (C) The time course experiment for the accumulation of phosphorylated p53 at Ser-15 after UV. 293 cells were exposed to UV at 20 J/m2 and harvested 0 to 72 hr after treatment as indicated. A Western blot analysis was performed using anti-phosphorylated p53 (Ser-15) antibody. Relative expression of phosphorylated p53 to whole p53 is shown. Data represent the means ± SD of three independent experiments. (D) The time course experiment of hRad9/p53 complex formation after exposure to UV. 293 cells were exposed to UV at 20 J/m2 and harvested 0 to 72 hr after treatment. Top, immunoprecipitation with anti-p53 antibody and Western blot analysis using anti-Rad9 antibody; middle, immunoprecipitation with anti-Rad9 antibody and Western blot with anti-p53 antibody; bottom, immunoprecipitation with anti-Rad9 antibody and Western blot with anti-phosphorylated p53 (Ser-15) antibody.