Figure 2: Proteasomal HSF1 degradation in HD is mediated by Phospho-S303/S307.
(a,b) PC12 cells expressing Htt-Q74 for 1, 2 or 3 days followed by Heat Shock (HS) and recovery as indicated. Control and HS in the absence of Dox correspond to cells incubated during 3 days at 37 °C. (c) Htt-Q74 cells Dox-induced or not for 3 days and exposed to 2 μM 17-AAG and/or MG132 (5 μM) for 6 h and extracts immunoblotted with the indicated antibodies. (d) Diagram of the effects of 17-AAG and MG132 treatment and Htt-Q74 expression in HSF1. (e) Htt-Q74 cells transfected with human influenza haemagglutinin-ubiquitin (HA-Ub) plasmid Dox induced or not and treated with 5 μM MG132 treatment for 6 h. Whole-cell extract and HA immunoprecipitated (IP:HA) and HSF1 immunoprecipitated samples (IP:HSF1) were immunoblotted as indicated. (f) Transcript levels for indicated E3 ligases evaluated by qRT-PCR from striatum of WT and KIQ175 mice at 6 months. Error bars represent±s.e.m., (n=3). Unpaired t-test, *P<0.05. (g) Human striatum samples from HD patients and controls and (h) Mouse striatum from 12 months old WT and KIQ175 mice were immunoblotted for HSF1 and Fbxw7. (i) Fbxw7 siRNA in STHdhQ7 and STHdhQ111 cells using scrambled RNA (Scr) as control. HSF1 was quantified using Quantity One image software and normalized using GAPDH as loading control and referenced to control at 37 °C in the non-pathogenic STHdhQ7. (j) hsf1−/− MEFs transfected with WT HSF1 or S303A mutant and Fbxw7-FLAG; samples immunoprecipitated with anti-FLAG and HSF1 detected. (k) hsf1−/− MEFs expressing Dox-inducible Htt-Q74-GFP transfected with WT HSF1 or HSF1-S303A HS and 1 h recovery at 37 °C for 7 h. HSF1 was quantified using Quantity One image software and normalized using GAPDH as loading control and referenced to control (−Dox) expressing WT HSF1. See Supplementary Fig. 9 for uncropped immunoblots.
