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. 2012 Oct 31;287(52):43984–43994. doi: 10.1074/jbc.M112.421545

FIGURE 7.

FIGURE 7.

RNF8-mediated ubiquitination of Nbs1 promotes DSB repair by HR. A, U2OS cells were stably integrated with EGFP-based repair substrates for HR and MMEJ (described previously (11)) and stably expressing shRNAs against RNF8 or control. I-SceI was induced by retroviral infection, and 7 days later, cells were collected for FACS analysis of EGFP-positive cells. The immunoblot shows silencing of RNF8 (*, nonspecific band), with Ku70 used as a loading control. B, U2OS cells carrying HR repair substrate were stably expressed with FLAG-RNF8 WT or the indicated mutants, with endogenous RNF8 silenced by RNF8sh. I-SceI was induced and FACS analysis was performed. Immunoblot shows expression of RNF8 variants, with Ku70 used as a loading control. C, U2OS cells carrying HR repair substrate were stably expressed with Nbs1-Myc WT or the indicated mutants, with endogenous Nbs1 silenced by Nbs1sh. I-SceI was induced, and FACS analysis was performed. Immunoblot shows expression of Nbs1 variants, with Ku70 used as a loading control. Error bars, S.D.