Effect of BRCA1 deficiency on the DNA damage-inducible association of FANCJ and RPA in BRCA1-deficient cells. (A) siRNA depletion of BRCA1. WCE of HeLa cells transfected with control or BRCA1 siRNA were probed using an antibody against BRCA1. Actin serves as a loading control. (B) WCEs were prepared from control siRNA or BRCA1 siRNA HeLa cells that were untreated or exposed to IR (10 Gy) or MMC (500 ng/mL) as indicated and immunoprecipitated with FANCJ antibody. The blot was probed with rabbit anti-FANCJ (top) and mouse anti-RPA70 (bottom) antibodies. (C) HCC 1937 cells form RPA but not FANCJ DNA damage-inducible foci. BRCA1 mutant HCC 1937 or reconstituted cells were treated with the DNA-damaging agent MMC (500 ng/mL) or exposed to 10 Gy IR as described in “Immunofluorescence cellular localization studies.” After fixation and permeabilization, cells were incubated with anti-RPA (green) and anti-FANCJ (red) antibodies. (D) Quantitation of FANCJ-RPA colocalizing foci as described in panel C. Percentages of RPA foci colocalizing with FANCJ are represented (□). Percentages of FANCJ foci colocalizing with RPA are represented (▩). Experimental data are the mean of at least 3 independent experiments with SD indicated by error bars.