Hsp90 stabilizes cytoplasmic FANCA in a FANCG-independent manner. (A) Cell fractionation study of HeLa/FH-FANCA. Cytoplasmic extracts (Cyto) and nuclear extracts (Nuc) prepared from control HeLa and HeLa/FH-FANCA cells were immunoprecipitated (IP) with anti-FLAG antibody followed by immunoblotting with the indicated antibodies (upper panels). The same extracts were directly immunoblotted with the indicated antibodies (lower panels). Topoisomerase II (Top II) and tubulin-β are nuclear and cytoplasmic markers, respectively. (B) Cell fractionation study of parental HeLa cells. Whole-cell lysates (Whole) and cytoplasmic extracts (Cyto) and nuclear extracts (Nuc), prepared from HeLa cells (1 ×10⁷ cells) were immunoprecipitated with either control rabbit IgG or anti-FANCA antibody, and then immunoblotted with anti-FANCA and anti-Hsp90 antibodies (upper panels). The same extracts were directly immunoblotted with the indicated antibodies (lower panels). Topoisomerase II (Top II) and tubulin-β are nuclear and cytoplasmic markers, respectively. (C) In vitro interaction of FANCA with Hsp90. In vitro transcription/translation reactions were programmed with empty vector (control), pcDNA3 Myc-FANCA (Myc-FANCA), or pcDNA3 Myc-FANCG (Myc-FANCG) in the absence (−) or presence (+) of 10 μM 17-AAG (lanes 1-4). Reaction mixtures were immunoprecipitated with anti-Myc antibody and immunoblotted with the indicated antibodies (upper panels), or immunoprecipitated with anti-Hsp90 antibody and immunoblotted with the indicated antibodies (middle panels). The arrow indicates Myc-FANCA. A portion (10%) of the input material was directly immunoblotted with anti-Myc antibody to detect synthesized FA proteins (lower panels). FANCG was synthesized alone or with Myc-FANCA, in the absence (−) or presence (+) of 10 μM 17-AAG, as described (lanes 5-7). Reaction mixtures were immunoprecipitated with anti-Myc antibody and immunoblotted with the indicated antibodies. (D) Recognition of different regions of FANCA by Hsp90 and FANCG. FANCA full-length protein and deletion mutants with a FLAG-tag at their N-termini were synthesized in vitro. Structures of the deletion mutants are schematically shown at the bottom of the figure. Reaction mixtures were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-Hsp90 antibodies. Arrows indicate synthesized FANCA polypeptides. The same FANCA polypeptides were cosynthesized with Myc-FANCG in vitro, and reaction mixtures were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-FLAG (data not shown) and anti-Myc antibodies. Results of binding studies are summarized on the right for each mutant (bottom, ranging from negative, −, to strongly positive, ++). (E) Interaction of Hsp90 with FANCA mutants. Lysates from GM6914 cells stably expressing FLAG-tagged wild-type (WT) or mutant FANCA proteins were immunoprecipitated with anti-FLAG antibody (upper panels) or anti-Hsp90 antibody (lower panels) and immunoblotted with the indicated antibodies. (F) Sensitivity of ΔNLS FANCA mutant to 17-AAG. GM6914 cells expressing wild-type FANCA (WT) or ΔNLS mutant were treated with 100 μg/mL CHX alone, (CHX) or with 250 nM 17-AAG (CHX + 17-AAG) for the indicated times. Cell lysates were immunoblotted with anti-FANCA and anti–tubulin-β antibodies. FANCA signals were quantified and normalized against tubulin-β signals. Data represent means ± SE from 3 independent experiments (bottom graph).