Hsp90 specifically associates with FANCA in vivo. (A) Coimmunoprecipitation of Hsp90 with FANCA. Lysates from control HeLa cells and HeLa/FH-FANCA cells were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted with anti-FLAG and anti-Hsp90 antibodies (lanes 1 and 2). Reciprocally, lysates from HeLa/FH-FANCA cells were immunoprecipitated with either control mouse IgG or mouse anti-Hsp90 monoclonal antibody and immunoblotted with anti-FLAG and anti-Hsp90 antibodies (lanes 3 and 4). Lysates from HeLa cells were immunoprecipitated with either control rabbit IgG or anti-FANCA and immunoblotted with the indicated antibodies (lanes 5 and 6), or immunoprecipitated with either control mouse IgG or anti-Hsp90 antibody and immunoblotted with the indicated antibodies (lanes 7 and 8). (B) Inhibition of the interaction between FANCA and Hsp90 by 17-AAG. HeLa/FH-FANCA cells were treated with vehicle (−) or 250 nM 17-AAG (+) for 15 minutes (lanes 2 and 3). Vehicle-treated parental HeLa cells were used as control (lane 1). Lysates from these cells were immunoprecipitated with anti-FLAG antibody and immunoblotted with the indicated antibodies (upper panels), or were immunoprecipitated with anti-Hsp90 antibody and immunoblotted with the indicated antibodies (middle panels). Lysates were directly immunoblotted with the indicated antibodies to confirm that FH-FANCA and Hsp90 protein levels were constant after 17-AAG treatment (lower panels). (C) Stabilization of the interaction between FANCA and Hsp90 by molybdate. HeLa/FH-FANCA cells were lysed in a buffer (10 mM HEPES, pH 7.5, 10 mM MgCl₂, 150 mM KCl, 0.2% Tween 20) in the presence (+) or absence (−) of 20 mM molybdate. Lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with the indicated antibodies (upper panels), or immunoprecipitated with anti-Hsp90 antibody and immunoblotted with the indicated antibodies (lower panels).