Iblot semi dry transfer apparatus

Cell lysates were prepared and quantified according to established methods. To each well of a 4–12 % or 12 % SDS-polyacrylamide gel, 15–30 μg total protein was applied, electrophoresed, and transferred to nitrocellulose membrane using an iBlot semi-dry transfer apparatus (Life Technologies). Membranes were blocked using Tris-buffered saline with 5 % nonfat milk (pH 7.6; Sigma). Primary antibodies used in this study were SDHA, SDHB, Histone H3 (Abcam), PRODH/POX, NDUFA10, Complex III Rieske FeS (Santa Cruz), Dimethyl Histone H3 (K4), Dimethyl Histone H3 (K36), COX IV (Cell Signaling, Danvers MA), β-actin (Novus Biologicals, Littleton, CO), and subsequently by a secondary anti-mouse/anti-rabbit IgG antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). All blots were washed in Tris-buffered saline with Tween 20 (pH 7.6; Sigma). Detection was done using an ECL kit (GE Healthcare, Pittsburgh, PA). Signals were quantified using Image Studio Light V5 (LI-COR Biosciences, Lincoln, NE).

Proteins were separated by SDS-PAGE using 3–8% Tris-acetate gels or 4–12% Tris-bis gels (Life Technologies) then transferred to nitrocellulose using iBlot semi-dry transfer apparatus (Life Technologies) or Trans-Blot Turbo (BioRad) and developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Statistical analysis was performed using Microsoft Excel or Prism (GraphPad Software). Pixel intensity quantification was performed using ImageJ (NIH) and signal for Huntingtin was standardized to signal for GAPDH. Some blots were stripped before re-probed using Reblot (Millipore) per manufacturer’s instructions. The stripping buffer is pH 13.3.