Effect of wild-type and phosphorylation-defective hRad9 mutants expression on P21 transactivation. (A) Western blots of lysates from 293 transfectants of wild-type or phosphorylation-defective RAD9 mutants. Lysates were analyzed by Western blotting, probed with anti-Rad9 (top), anti-FLAG (middle), or anti-actin (bottom) antibody. (B) P21 transcription assay with wild-type and phosphorylation-defective RAD9. mutants using P21 promoter-luciferase reporter system. After transfection into 293 cells, the relative luciferase activities are shown as the ratios of Firefly to Renille. Data represent the means ± SD of four independent experiments.