Figure 4.
UBC13-Mediated Ubiquitin Signaling Involving UBC13, RAP80, and BRCA1 is Required for Efficient Recruitment of MRE11 Nuclease onto DSB Sites in G1 Cells
(A) Quantification of MRE11-positive MCF-7 cells with at least 10 foci per nucleus for the indicated genotypes. Serum-starved MCF-7 cells were treated with etoposide (10 μM) for 30 min. Error bars were plotted for standard deviation (SD) from three independent experiments. Single, double, and triple asterisks indicate p = 2.3 × 10−4, p = 5.3 × 10−5, and p = 1.3 × 10−4, respectively, calculated by Student's t test. Representative images of etoposide-induced MRE11/53BP1 foci are shown in Figure S4.
(B) Etoposide-induced complex formation of BRCA1 and MRE11 in G1-phase MCF-7 cells. Whole-cell extracts (WCEs) were prepared from the indicated cells, untreated (“-”) or treated (“+”) with etoposide (10 μM) for 0.5 h. “Input” indicates 5% of WCEs used for immunoprecipitation. BRCA1 was immunoprecipitated from the WCEs. Intensities of the immunoprecipitated MRE11 bands for the indicated genotypes were normalized to those of the input. The graph indicates relative band intensities of the MRE11 bands in comparison with the untreated wild-type (Lane 1). Single and double asterisks indicate p = 2.1 × 10−2 and p = 1.5 × 10−3, respectively, calculated by Student's t test.