Figure 3
Figure 3. 17-AAG induces degradation of FANCA via the ubiquitin-proteasome pathway. (A-B) FANCA degradation induced by 17-AAG. HeLa/FH-FANCA (A) and parental HeLa (B) cells 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 the indicated antibodies (upper blots). FANCA signals were quantified and normalized against tubulin-β signals. Data represent means ± SE from 3 independent experiments (bottom graphs). (C) Proteasome inhibitors block 17-AAG–induced FANCA down-regulation. HeLa/FH-FANCA cells were treated with 17-AAG or MG132, at appropriate concentrations for 4 hours (lanes 1-6). Cell lysates prepared using SDS-sample buffer were immunoblotted with anti-FLAG and anti–tubulin-β antibodies. HeLa cells were treated with 17-AAG and proteasome inhibitors, MG132 or lactacystin, at appropriate concentrations for 4 hours (lanes 7-15). Cell lysates prepared using SDS-sample buffer were immunoblotted with anti-FANCA and anti–tubulin-β antibodies. (D) Enhancement of polyubiquitination of FANCA by 17-AAG. HeLa/FH-FANCA cells were treated with vehicle (−) or 250 nM 17-AAG (+), in the absence (−) or presence (+) of 10 μM MG132 for 1 hour (lanes 1-4). Cell lysates prepared using ubiquitin lysis buffer were immunoprecipitated with anti-FLAG antibody and immunoblotted with antiubiquitin and anti-HA antibodies to detect polyubiquitinated FANCA (Ubn-FANCA). The arrow indicates nonubiquitinated FANCA. HeLa cells were transfected with empty vector (−; lane 5) or a plasmid encoding HA-ubiquitin (HA-Ub; +; lanes 6-10). After 24 hours of transfection, cells were treated with 17-AAG and MG132, as described. Cell lysates were immunoprecipitated with either anti-FANCA antibody (lanes 5-9) or control rabbit IgG (lane 10) and immunoblotted with anti-HA and anti-FANCA antibodies. (E) Association of CHIP with FANCA. HeLa/FH-FANCA cells were treated with 17-AAG and MG132 alone or in combination (lanes 1-4), as described in Figure 3D. Cell lysates were immunoprecipitated using anti-FLAG M2 agarose and immunoblotted with the indicated antibodies (upper panels). The same lysates were immunoblotted with the indicated antibodies (lower panels).

17-AAG induces degradation of FANCA via the ubiquitin-proteasome pathway. (A-B) FANCA degradation induced by 17-AAG. HeLa/FH-FANCA (A) and parental HeLa (B) cells 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 the indicated antibodies (upper blots). FANCA signals were quantified and normalized against tubulin-β signals. Data represent means ± SE from 3 independent experiments (bottom graphs). (C) Proteasome inhibitors block 17-AAG–induced FANCA down-regulation. HeLa/FH-FANCA cells were treated with 17-AAG or MG132, at appropriate concentrations for 4 hours (lanes 1-6). Cell lysates prepared using SDS-sample buffer were immunoblotted with anti-FLAG and anti–tubulin-β antibodies. HeLa cells were treated with 17-AAG and proteasome inhibitors, MG132 or lactacystin, at appropriate concentrations for 4 hours (lanes 7-15). Cell lysates prepared using SDS-sample buffer were immunoblotted with anti-FANCA and anti–tubulin-β antibodies. (D) Enhancement of polyubiquitination of FANCA by 17-AAG. HeLa/FH-FANCA cells were treated with vehicle (−) or 250 nM 17-AAG (+), in the absence (−) or presence (+) of 10 μM MG132 for 1 hour (lanes 1-4). Cell lysates prepared using ubiquitin lysis buffer were immunoprecipitated with anti-FLAG antibody and immunoblotted with antiubiquitin and anti-HA antibodies to detect polyubiquitinated FANCA (Ubn-FANCA). The arrow indicates nonubiquitinated FANCA. HeLa cells were transfected with empty vector (−; lane 5) or a plasmid encoding HA-ubiquitin (HA-Ub; +; lanes 6-10). After 24 hours of transfection, cells were treated with 17-AAG and MG132, as described. Cell lysates were immunoprecipitated with either anti-FANCA antibody (lanes 5-9) or control rabbit IgG (lane 10) and immunoblotted with anti-HA and anti-FANCA antibodies. (E) Association of CHIP with FANCA. HeLa/FH-FANCA cells were treated with 17-AAG and MG132 alone or in combination (lanes 1-4), as described in Figure 3D. Cell lysates were immunoprecipitated using anti-FLAG M2 agarose and immunoblotted with the indicated antibodies (upper panels). The same lysates were immunoblotted with the indicated antibodies (lower panels).

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