17-AAG induces a rapid down-regulation of FANCA. HeLa/FH-FANCA (A) and parental HeLa (B) cells were treated with 250 nM 17-AAG for the indicated times. Lysates were immunoblotted with the indicated antibodies. To assess total cellular levels of FANCM, whole-cell lysates were immunoprecipitated (IP) and immunoblotted with anti-FANCM antibody. FA protein signals were quantified and normalized against tubulin-β signals. Data represent means ± SE from 3 independent experiments (bottom graphs). (C) Dose-dependent down-regulation of FANCA by 17-AAG. HeLa and MCF7 cells were treated with various concentrations of 17-AAG for 2 hours. Cell lysates were immunoblotted with the indicated antibodies (upper panels). FANCA signals were quantified and normalized against tubulin-β signals. Data represent means ± SD from 3 independent experiments (bottom graphs). (D) After treatment with vehicle (−) or 17-AAG (+), lysates from K562 cells (500 nM, 4 hours), Jurkat cells (250 nM, 2 hours), RPMI8226 cells (1 μM, 2 hours), and NIH3T3 cells (1 μM, 4 hours) were immunoblotted with appropriate antibodies (lanes 1-8). HeLa cells were treated with 250 nM 17-AAG, 2 μM radicicol, or 2 mM novobiocin for 2 hours (lanes 9-12). Cell lysates were immunoblotted with anti-FANCA and anti–tubulin-β antibodies. Numbers at the bottom of sample lanes 9-12 represent relative FANCA protein levels normalized against control.