p400 is required for RNF8-dependent ubiquitination of the chromatin. (a) 293T cells expressing p400 or p400^ATPase were irradiated as indicated. Cells were fixed and processed by immunofluorescent staining using FK2 antibody. Bar, 5 µm. (b) 293T cells expressing p400 or p400^ATPase were irradiated (2Gy). Immunofluorescent staining using the FK2 antibody was used to detect proteins ubiquitinated by RNF8. Cells with >5 foci per cell were counted, with an average of 100 cells per slide. Results ± SE (n = 100). (c) 293T cells expressing p400 or p400^ATPase were transiently transfected with vector or p84-ZFN. 18 h later, ChIP assays using the FK2 antibody and primer pairs located at −3.5 kb, −1.5 kb, −0.5 kb, 0.5 kb, 1.5 kb, and 3.5 kb were performed. Results ± SE (n = 3). (d) 293T cells expressing p400 or p400^ATPase were irradiated (2Gy), fixed, and immunofluorescent staining to detect RNF8 and FK2 performed. Bar, 5 µm. (e) Quantitation of FK2 and RNF8 foci in p400 and p400^ATPase cells from d. Cells with >5 foci were counted, with an average of 100 cells per slide counted. Experiments represent at least three independent replicates. Results ± SE (n = 100).