Donald J. representative experiment. (not significant) was determined by Student’s test. and display cleaved caspase-3-positive cells (show significant variations (**, 0.01; *, 0.05; test. In each experiment, 156266 cells were counted. indicate the significant difference (**, 0.01; test. The results of no treatment (indicate the significant difference (**, 0.01; test. In each experiment, 35128 cells were counted. indicate significant variations (**, 0.01; *, 0.05; test. In each experiment, Schisandrin B 39249 cells were counted. A recent study showed that NIH3T3 cells transformed by expression of a temperature-sensitive v-Src mutant were strongly resistant to a variety of apoptosis inducers, including UV light (29). To examine the involvement of Src-family tyrosine kinases in UV-induced apoptosis, we treated parental HeLa S3 cells and HeLa S3/shKu70 cells with UV light in the presence or absence of PP2 or SU6656, another Src inhibitor, and found that Src inhibition improved the level of apoptosis in parental HeLa S3 cells prominently at a low dose of UV (Fig. 4and and show the significant difference (*, 0.05) calculated by Student’s test. The results of no treatment (and indicate significant variations (**, 0.01; *, 0.05; test. The results of no treatment (indicate the significant difference (***, 0.001; test. indicates the significant difference (*, 0.05; test. indicate the significant difference (**, 0.01; test. indicate the significant difference (*, 0.05) calculated by Student’s test. indicates the significant difference (**, 0.01; test. indicate the significant difference (*, 0.05) calculated by Student’s test. indicates nonspecific bands. The percentage of cells exhibiting cleaved caspase-3 was quantitated. Results (%) represent the means S.D. from three self-employed experiments. indicate the significant difference (**, 0.01) calculated by Student’s test. The result of vector only is the means from two self-employed experiments. In each experiment, 4980 cells were counted. Next, apoptosis can be initiated by activation of the proapoptotic element Bax (54, 55), and Ku70 suppresses Bax-mediated apoptosis by sequestering Bax away from mitochondria through the Ku70-Bax connection (43,C48). Cotransfection of Bax with control vector, c-Src, or v-Src, a highly triggered variant of c-Src, showed the levels of Bax-mediated apoptosis were decreased by manifestation of c-Src and v-Src (Fig. 7and and shows the significant difference (*, 0.05) calculated by Student’s test. indicates the significant difference (*, 0.05) calculated by Student’s test. and indicate the significant difference (*, 0.05; test. and kinase assays, we verify direct phosphorylation of Ku70 by Src (Fig. 1, and and and and and and kinase assays were performed as explained (13, 14, 84, 87). In brief, c-Src was immunoprecipitated with anti-HA antibody from Triton X-100 lysates of COS-1 Schisandrin B cells transfected with c-Src (c-Src-HA) or c-Src(KD) (c-Src(KD)-HA). myc-Ku70-WT was immunoprecipitated with anti-myc antibody from Triton X-100 lysates of COS-1 cells transfected with myc-Ku70-WT. After washing, myc-Ku70-WT was eluted with 0.2 m glycine-HCl buffer, pH 2.5, and immediately neutralized Mouse monoclonal to Mcherry Tag. mCherry is an engineered derivative of one of a family of proteins originally isolated from Cnidarians,jelly fish,sea anemones and corals). The mCherry protein was derived ruom DsRed,ared fluorescent protein from socalled disc corals of the genus Discosoma. Schisandrin B with 1 m Tris-HCl, pH 8.8. Equivalent amounts of c-Src immunoprecipitates bound to the beads were reacted with eluted myc-Ku70-WT in kinase buffer (40 mm HEPES, pH 7.4, 0.1% Triton X-100, 5 mm MnCl2, 5 mm MgCl2, and 1 mm Na3VO4) containing 100 m Schisandrin B unlabeled ATP at 30 C for the indicated periods. Phosphorylated bands were immunodetected with anti-Tyr(P) antibody, and the intensity of chemiluminescence was measured using Amount One software (Bio-Rad). Composite numbers were prepared using GIMP 2.6.2 and Illustrator 16.0. Author Contributions M. M. and Na. Y. conceived of the study, designed the experiments, and published the manuscript. M. M. carried out the majority of experiments. S. K., T. T., and T. K. performed phosphoproteomic analysis. S. K..