Figure 1.
MSH6 interacts with Ku70. (A) HeLa cells were untreated (UT) or treated with 100 ng/ml NCS or 5 Gy ionizing radiation (IR). Proteins were immunoprecipitated (IP) from the lysates using an anti-MSH6 antibody 3 h after treatment. Immunoprecipitates were then subjected to western-blot analysis using antibodies specific for Ku70 or MSH6. The fourth lane contain 5% input for Ku70 and 20% input for MSH6. Normal rabbit IgG was used for negative control immunoprecipitations. (B) HeLa cells were untreated or treated with 100 ng/ml NCS for 3 h. Proteins were immunoprecipitated from the lysates using an anti-Ku70 antibody. Immunoprecipitates were then subjected to western-blot analysis using antibodies specific for MSH6, MSH2 or Ku70. The third and fourth lane contains 5% input for Ku70 and 20% input for MSH6 and MSH2. Normal rabbit IgG was used for negative control immunoprecipitations. (C) HeLa cells were untreated or treated with 100 ng/ml NCS for 3 h. Proteins were immunoprecipitated from the lysates using an anti-MSH6 antibody. Immunoprecipitates were then subjected to western-blot analysis using antibodies specific for Ku70, Ku86, DNA-PKCS, XRCC4 or MSH6. The third and fourth lane contains 5% input for Ku70 and 20% input for Ku86, DNA-PKcs, XRCC4 and MSH6. Normal rabbit IgG was used for negative control immunoprecipitations. (D) HeLa cells were untreated or treated with 100 ng/ml NCS for 3 h. Whole cell lysates were treated with 50 µg/ml ethidium bromide (EtBr) on ice for 30 min (lane 3) or 100 µg/ml DNase I for 20 min at 37°C (lane 4) or mock-treated (lane 2). Cell lysates were then subjected to coimmunoprecipitation assays with MSH6 antibody and detected by anti-Ku70 or anti-MSH6. The fifth lane contains 5% input for Ku70 and 20% input for MSH6. (E) HeLa cells were untreated or treated with 100 ng/ml NCS, and cells were lysed at the indicated times. Whole-cell lysates were subjected to immunoprecipitation using an anti-MSH6 antibody, and the resulting immunoprecipitates were subjected to western-blot analysis using anti-Ku70 and anti-MSH6 antibodies. Graphs show the quantification of the level of Ku70. The sixth lane contains 5% input for Ku70 and 20% input for MSH6. The data were normalized to the untreated control (as the value of 1) and are the mean ± SD of three independent experiments. (F) HEK293T cells were transfected with full-length MSH6 and HA-tagged Ku70 expression vectors. After 24 h, cells were untreated or treated with 100 ng/ml NCS for 3 h. Proteins were immunoprecipitated from HEK293T cell lysates using an anti-MSH6 antibody, and the resulting immunoprecipitates were subjected to western-blot analysis using anti-HA or anti-MSH6 antibodies. Graphs show the quantification of the level of HA-Ku70. The third and fourth lane contains 20% input. The data were normalized to the untreated control (as the value of 1) and are the mean ± SD of three independent experiments.