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. 2010 Oct 4;191(1):31–43. doi: 10.1083/jcb.201001160

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© 2010 Xu et al.

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Figure 7. — Recruitment of p400 to DSBs requires mdc1. (a) H2AX^−/− MEFs or H2AX^−/− MEFs complemented with H2AX (H2AX^wt) were exposed to 5 µM bleomycin. 20 µM Wortmannin was added 60 min before bleomycin. Cells were fractionated in 1.0 M NaCl, and released histones detected by Western blot. (b) 293T cells expressing vector or mdc1 shRNA (shmdc1) were exposed to 5 µM bleomycin. Cells were fractionated in 1.0 M NaCl, and released histones detected by Western blot. (c) 293T cells expressing GFP or mdc1 shRNA were transiently transfected with vector or p84-ZFN. 18 h later, ChIP assays using p400 antibody and primer pairs located at +1.5 kb were performed. Results ± SE (n = 3). (d) 293T cells stably expressing FLAG-RNF8 or RNF8 lacking the catalytic domain (FLAG-RNF8^δring), which functions as a dominant-negative inhibitor of endogenous RNF8 function (Huen et al., 2007), were exposed to bleomycin. Cells were fractionated in 1.0 M NaCl, and released histones detected by Western blot.