Transfer apparatus

Tissues (lungs, SCAT and EWAT) were homogenized in lysis buffer supplemented with protease inhibitors (cOmplete^TM Protease Inhibitor Cocktail, Sigma-Aldrich). Whole tissue protein extracts (40–50 µg) were fractionated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes using a transfer apparatus as per the manufacturer’s instructions (Life Technologies). Membranes were blocked with 5% bovine serum albumin (BSA) in PBS/0.1% Tween for 1 h, washed and probed overnight at 4 °C with polyclonal antibody against UCP1 (1:500) (Abcam, Cambridge, UK). Membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (KPL, Milford, MA, USA) for 2 h. After three washings, blots were developed with the ECL system (Bio-Rad Laboratories) according to the provided protocol. GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA) was used as protein loading control.

Total protein levels of lysates obtained from HEK293T cells and Drosophila HD model, the latter produced as in ref. 9 (link), were quantified using the Pierce BCA Protein Assay kit (Thermo Scientific) according to manufacturer’s protocol. 20 µg of total lysates were denatured at 95 °C in 4x loading buffer (125 mM TrisHCl pH 6.8, 6% SDS, 4 M urea, 4 mM EDTA, 30% glycerol, 4% β-mercaptoethanol and Bromophenol blue) and loaded on NuPAGE 4–12% Bis-Tris Gel or WedgeWell 6% Tris-Glycine Gel (Life Technologies). Proteins were transferred on PVDF membranes using the transfer apparatus from Life Technologies or Trans-Blot Turbo Transfer System from Biorad following manufacturer’s protocol. Membranes were stained for 30 minutes in TBS, 0.1% Tween, 0.4% PFA, before 1 hour blocking in TBS, 0.1% Tween, 5% not fat milk. Incubations with primary antibodies were performed overnight at 4 °C. Incubations with secondary anti-mouse- or rabbit horseradish peroxidase conjugated antibodies were carried out for 1 hour at room temperature. Protein bands were revealed using chemiluminescence substrate (ECL from Life Technologies) and images were acquired using a Chemidoc imager (Biorad). Similarly, synthetic huntingtin exon 1 proteins were loaded on NuPAGE 4–12% Bis-Tris Gel (Life Technologies) for Western blotting analysis.

Cultured cells were solubilized in lysis buffer (10 mM Tris-HCl [pH7.2], 150 mM NaCl, 0.5% deoxycholic acid sodium salt, 0.1% Nonidet P-40, 5 mM EDTA, and proteinase inhibitor cocktail (Sigma) on ice and then centrifuged (14,000 r.p.m) for 15 min at 4°C. The supernatant was mixed with 4 × protein sample buffer (4 × NuPAGE® LDS Sample Buffer [Life Technologies, NY, USA], 3% 2-mercaptoethanol) and heated for 10 min at 98°C. The proteins were then separated by electrophoresis on 4%-12% NuPAGE® Bis-Tris Mini Gels (Life Technologies, CA USA) and transferred to Protoran− Nitrocellulose membranes (GE Healthcare Bio-Sciences, CA, USA) in a transfer apparatus (Life Technologies). The membranes were blocked with 5% skim milk and 0.1% Tween in Tris-buffered saline (TBS). Immunodetection was done utilizing the primary antibodies; MBD4 (Sigma), β-tubline (GE Healthcare Bio-Sciences), and horseradish peroxidase (HRP) linked F(ab’)2 secondary rabbit or mouse antibodies (Santa Cruz Biotechnology, CA, USA). The signals were detected using an LAS-4000 luminescent image analyzer (GE Healthcare Bio-Sciences) utilizing a chemiluminescent solution.