Precast 4 15 bis tris gels

The brain samples were homogenized in Soluble lysis buffer (10 mM Tris pH 7.4, 1% Triton-X 100, 150 mM NaCl, 10% glycerol), douncing slowly 30 times in a 1 mL glass douncer. The homogenate was transferred into labeled, ”Insoluble“ tubes and lyse on ice for 1 h. The samples were centrifuged at 4 °C at 15,000×g for 20 min. The supernatant was recovered as the ”Soluble“ fraction. The pellet was washed with 500 $\mathsf{\mu }$ L of lysis buffer (×2) and centrifuged at 4 °C at 15,000×g for 5 min after each wash. Then, the pellet was resuspended with 150 $\mathsf{\mu }$ L of lysis buffer supplemented with 4% SDS and sonicated each sample for 30 s at room temperature with a probe sonicator at 40% of amplitude (Insoluble fraction). Finally, the insoluble fraction was boiled for 30 min. Protein concentrations were estimated using the BCA assay (Pierce). SDS-PAGE was performed using Precast 4–15 Bis-Tris gels (Bio-Rad). Gels were run in a 1× tris-acetate SDS running buffer for 2 h and transferred to a 0.45 $\mathsf{\mu }$ m PVDF membrane using the Turbo Trans-Blot transfer system (Bio-Rad) for 30 min. Membranes were blocked with 5% skim milk in PBS with 0.1% Tween 20 (PBS-T) for 1 h at room temperature and incubated with reconstituted anti-HTT (MAB5374, 1:1000), and anti-b-actin HRP (sc-47778 1:5000) primary antibodies in 5% skim milk overnight at 4 °C.

The right cerebral hemisphere was dissected into the different areas of the brain to be analyzed. The tissues were immediately transferred to dry ice and stored at −80 °C for further analysis. The brain samples were homogenized in RIPA with 1% SDS and a protease inhibitor cocktail (1 tablet per 10 mL, Roche complete, EDTA-free, Sigma-Aldrich) using a sonicator with a 10 s pulse at 40% amplitude. Protein concentrations were estimated using the BCA assay (Pierce, Appleton, WI, USA). Cell lysates was denatured under reducing conditions by boiling 30 $\mathsf{\mu }$ g of total protein with 1 M DTT and 4× LDS sample buffer (Invitrogen) at 95 °C for 5 min. SDS-PAGE was performed using Precast 4–15% Bis-Tris gels (Bio-Rad, Hercules, CA, USA). Gels were run in 1× tris-acetate SDS running buffer for 2 h and transferred to a 0.45 $\mathsf{\mu }$ m PVDF membrane using the Turbo Trans-Blot transfer system (Bio-Rad) for 30 min. Membranes were blocked with 5% skim milk in PBS with 0.1% Tween 20 (PBS-T) for 1 h at room temperature and incubated with reconstituted anti-HTT (MAB5374, 1:1000) and anti-b-actin HRP (sc-47778 1:5000) primary antibodies in 5% skim milk overnight at 4 °C.