Disruption of HSP90 function induces PABPN1 degradation.
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(A) Reduction of A17-PABPN1 in 17-AAG-treated muscle cells. C2C12 myoblasts were transfected with HA-tagged A17-PABPN1 constructs. Twenty-four hours post-transfection, cells were treated with CHX (10 μg/ml) alone or together with 17-AAG (2 μM) or DMSO (2 μM) for the indicated times at 37°C. Lysates were blotted to show the expression of the proteins of interest. Band density was quantified and is shown in the line graph (bottom panel). Data are shown as the mean ± SEM (n = 5); **, P < 0.01. (B) Real-time PCR for mutant A17-PABPN1 mRNA. The mRNA levels of mutant A17-PABPN1 were similar with or without 17-AAG treatment. (C, D) Degradation of PABPN1 in 17-AAG treated myotubes cultured at 40°C. C2C12 myoblasts were transfected with GFP-tagged A10-PABPN1 and A17-PABPN1, and the cells were chased at 40°C in the presence of 17-AAG (2 μM) and CHX (10 μg/ml) for the indicated times 24 hr post-transfection. Lysates were blotted to show the expression of the proteins of interest. Band density was quantified and is shown in the line graphs (bottom panels). Values are mean ± SEM (n = 5). (E) 17-AAG reduced PABPN1 levels by 65% in cells transfected with non-silencing shRNA, whereas the suppression of HSP90 expression abrogated the 17-AAG-mediated reductions in PABPN1 expression. (F). The disruption of A17-PABPN1/HSP90 complex by 17-AAG. HEK293 cells were transfected with A17-PABPN1, and the cells were treated with 2 μM 17-AAG for the indicated times 36 hr post-transfection. Cell lysates were used for immunoprecipitation and blotting with the indicated antibodies. Lysates were also blotted to show the expression of the proteins of interest (bottom panels). (G) The reduction of PABPN1 by 17-AAG was inhibited by MG-132. C2C12 cells were transfected with A17-PABPN1. Thirty-six hours post-transfection, the cells were treated with vehicle (DMSO) or 17-AAG (2 μM) for 8 hr. Lysates were subjected to blotting with the indicated antibodies.