FANCJ and RPA are associated with each other in vivo and directly interact. (A) Coimmunoprecipitation of RPA with FANCJ. FANCJ antibody coprecipitates FANCJ and RPA from HeLa or FA-J–corrected cells but not from the FA-J extracts. The blot was probed with rabbit anti-FANCJ (top) and mouse anti-RPA (bottom) antibodies. (Lane 1) HeLa nuclear extract (15% of input), (lane 2) control immunoprecipitate from HeLa nuclear extract using normal rabbit IgG, (lane 3) immunoprecipitate from HeLa nuclear extract using rabbit anti-FANCJ antibody, (lane 4) FA-J whole-cell extract (WCE; 15% of input), (lane 5) immunoprecipitate from FA-J WCE using rabbit anti-FANCJ antibody, (lane 6) control precipitate from FA-J WCE using normal rabbit IgG, (lane 7) WCE from HeLa included as control for Western detection of FANCJ and RPA, (lane 8) FA-J corrected WCE (15% of input), (lane 9), immunoprecipitate from FA-J–corrected WCE using rabbit anti-FANCJ antibody, and (lane 10) control immunoprecipitate from FA-J–corrected WCE using normal rabbit IgG. (B) Coimmunoprecipitation of BRCA1 with FANCJ. FANCJ antibody coprecipitates FANCJ and BRCA1 from HeLa nuclear extract. The blot was probed with rabbit anti-FANCJ (top) and mouse anti-BRCA1 (bottom) antibodies. (Lane 1) HeLa nuclear extract (15% of input), (lane 2) immunoprecipitate from HeLa nuclear extract using rabbit anti-FANCJ antibody, and (lane 3) control immunoprecipitate from HeLa nuclear extract using normal rabbit IgG. (C) FANCJ antibody coprecipitates FANCJ and RPA from HeLa nuclear extracts in the presence of ethidium bromide or DNaseI. The blot was probed with rabbit anti-FANCJ (top) and mouse anti-RPA (bottom) antibodies. (Lane 1) HeLa nuclear extract (15% of input), (lane 2) immunoprecipitate from HeLa nuclear extract using rabbit anti-FANCJ antibody, (lanes 3 and 4) immunoprecipitate from HeLa nuclear extracts in the presence of 2 μg/mL DNaseI or 10 μg/mL ethidium bromide using rabbit anti-FANCJ antibody, and (lane 5) control immunoprecipitate from HeLa nuclear extract using normal rabbit IgG. (D,E) FANCJ and RPA form a complex by direct physical interaction. (D) RPA (96 nM heterotrimer, □) or ESSB (96 nM homotetramer, ■) was coated onto the ELISA plate. After blocking with 3% BSA, the wells were incubated with increasing concentrations of purified recombinant FANCJ protein (0-150 nM) for 60 minutes at 37°C. Wells were aspirated and washed 3 times, and bound FANCJ-WT protein was detected by ELISA with a rabbit polyclonal antibody against FANCJ. (E) Same as described for panel D except 2 μg/mL DNase I or 10 μg/mL ethidium bromide (EtBr) were incubated with RPA (96 nM) and FANCJ (77 nM) during the binding step in the corresponding wells. The values represent the mean of 3 independent experiments performed in duplicate with standard deviation (SD) indicated by error bars. (F) FANCJ and RPA interact by the 70-kDa subunit of RPA. Purified RPA, ESSB, and BSA (as indicated above the lanes) were subjected to SDS–polyacrylamide gel electrophoresis on 3 identical gels. The proteins bound to membranes were stained with Ponceau S or transferred to nitrocellulose membrane and incubated with either purified FANCJ (+FANCJ) or no protein (−FANCJ). Western blotting with anti-FANCJ antibody was then used to detect the presence of FANCJ on each membrane. The positions of the 70-, 32-, and 14-kDa subunits of RPA are indicated by asterisks. The positions of the molecular mass standards running parallel are shown on the left.