Western Blot Analysis of HTT Protein

Western blot analysis for HTT protein expression isolated from cell culture was performed as previously described.²⁴ (link) Briefly, 30 μg of total protein was separated on a Tris-acetate SDS-polyacrylamide gel (1.5 cm, 4% stacking gel/4.5 cm, 5% resolving gel, acrylamide:bis-acrylamide ratio of 49:1) in XT Tricine buffer (Bio-Rad) at 135 V in an ice-water bath. For proteins isolated from mouse brains, NuPAGE Tris-Acetate 3%–8% Protein Gel (Thermo Fisher Scientific) in NuPAGE Tris-Acetate SDS Running Buffer (Thermo Fisher Scientific) were used. After electrophoresis, the proteins were transferred overnight to a nitrocellulose membrane (Sigma-Aldrich) by the wet transfer method. The primary and secondary antibodies were used in PBS/0.1% Tween 20 buffer containing 5% nonfat milk. Immunoreactions were detected using Western Bright Quantum HRP Substrate (Advansta, Menlo Park, CA). Protein bands were scanned directly from the membrane using a camera, and band densities were quantified using a Gel-Pro Analyzer (Media Cybernetics). Plectin or calnexin was used as the reference protein. A list of all antibodies used is provided in Table S4.