Journal: Nature Communications

Article Title: Hsp70 and Hsp40 inhibit an inter-domain interaction necessary for transcriptional activity in the androgen receptor

doi: 10.1038/s41467-019-11594-y

Figure Lengend Snippet: Hsp70-targeted small molecules increase ubiquitination and degradation of polyQ-AR. a PC12 cells were transiently transfected to express HA-ubiquitin and CHIP, and AR112Q expression was induced for 48 h. Cells were treated with the Hsp70 modulator, JG-98 (0.5 μM) for the last 24 h and with 10 μM MG132 for the last 16 h. AR112Q was immuno-precipitated from lysates and then probed for ubiquitin (HA). Left: input. Middle: pull down. Right: quantification from three independent experiments. Data are mean ± S.E.M. from three independent experiments. * p < 0.05 by two-tailed t -test. b JG-98 promotes AR112Q degradation by the proteasome. PC12 cells were induced to express AR112Q for 48 h in the presence of R1881 (10 nM) and JG-98 (0.5 or 1.0 μM). Indicated samples were treated with lactacystine (10 μM) for the final 16 h. AR was detected by western blot. GAPDH controls for loading. c JG-98 does not induce a stress response. PC12 cells were induced to express AR112Q in the presence of R1881 (10 nM) for 48 h in the presence of 17-AAG (5 μM), JG-98 (0.5 μM) or vehicle control. Cell lysates were probed for Hsp25, Hsp40, Hsp70, Hsp90, Akt, and ERK1/2. GAPDH controls for loading. d PC12 cells were induced to express AR112Q in the presence of R1881 for 48 h, and treated with 17-AAG (1 µM) and/or JG-98 (1 µM), as indicated. Lysates were analyzed for AR by filter trap assay. Data are mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA. Source data for the quantifications shown in a , d are available as Source Data file

Article Snippet: Additional primary antibodies were used against Akt (C6E7; Cell Signaling Technology), ERK1/2 (9102, Signaling Technology), GAPDH (NB-600-502; Novus Biologicals), HSP90 (H114, sc7947; Santa Cruz Biotechnology), CHIP (PA5-32046, Thermo Scientific, Pierce), HA (MMS-101R, Covance, Biolegend), HSP25 (ADI-SPA-801-488; Enzo Life Science), and HSP70 (ADI-SPA-812; Enzo Life Science).

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Two Tailed Test, Western Blot, Control, TRAP Assay

Journal: Nature Communications

Article Title: Hsp70 and Hsp40 inhibit an inter-domain interaction necessary for transcriptional activity in the androgen receptor

doi: 10.1038/s41467-019-11594-y

Figure Lengend Snippet: JG-294 promotes clearance of AR113Q in mouse skeletal muscle. Three-month-old AR113Q males were injected i.p. with JG-294 (3 mg kg −1 ), 17-DMAG (10 mg kg −1 ), or vehicle every other day for two weeks. a Protein lysates from quadriceps were analysed by western blot for AR and GAPDH. b Quantification of AR monomer and AR high molecular weight species by densitometry. Data are mean ± S.E.M. from three mice per group. ** p < 0.01 by two-tailed t -test. V, Vehicle; JG, JG-294. c Relative expression of AR mRNA was determined by qPCR. Data are mean ± S.E.M. from three mice per group. d Quadriceps muscle protein lysates were analyzed by agarose gel electrophoresis for resolution of aggregates (AGERA) followed by western blot for AR. e Quadriceps were stained for AR (red) and analyzed by immunofluorescence microscopy. Nuclei were stained by DAPI (blue). Scale bar = 100 μm. At right, Quantification of AR signal within nuclei. Data are mean ± S.E.M. from three independent experiments. *** P < 0.0001 by two-tailed t -test. f Protein lysates from quadriceps were probed by western blot for Hsp70, Erk1/2 and GAPDH. Source data for the quantifications shown in panels b , c , and e are available as Source Data file

Article Snippet: Additional primary antibodies were used against Akt (C6E7; Cell Signaling Technology), ERK1/2 (9102, Signaling Technology), GAPDH (NB-600-502; Novus Biologicals), HSP90 (H114, sc7947; Santa Cruz Biotechnology), CHIP (PA5-32046, Thermo Scientific, Pierce), HA (MMS-101R, Covance, Biolegend), HSP25 (ADI-SPA-801-488; Enzo Life Science), and HSP70 (ADI-SPA-812; Enzo Life Science).

Techniques: Injection, Western Blot, High Molecular Weight, Two Tailed Test, Expressing, Agarose Gel Electrophoresis, Staining, Immunofluorescence, Microscopy