Figure 3.

BRCA2 depletion results in increased accumulation of pRPA32 after stalled fork–inducing DNA damage. (A and B) IF analysis and graph of RPA32 recruitment in U2OS cells transfected with control siRNA (siLuc) and siRNA for BRCA2. Cells were treated with 5 mM HU for 4 h and harvested right after or 20 h after treatment. Scale bars in A indicate 10 µm. (C) Western blot analysis of whole-cell lysate served as input for IF. (D and E) IF analysis and graph of RPA32 recruitment in U2OS cells transfected with control siRNA (siLuc) or siRNA for BRCA1, BRCA2, or RAD51. Cells were fixed 3, 8, and 24 h after UV damage (30 J/m²). **, P value < 0.05. Statistical significance was determined by the two-tailed Student’s t test and the error bars indicate SD (n = 3). Scale bars in D indicate 10 µm. (F) Western blot analysis of RPA32 accumulation in U2OS cells transfected with control siRNA (siLuc) or siRNA for RAD51. Cells were treated with 5 mM HU and harvested 3 h after treatment. Nuclear extracts were prepared.