Western Blot Analysis of Huntingtin Protein

The western blot analysis for the HTT protein (17/68Q tract) was performed as previously described (Fiszer et al., 2011 (link)). Briefly, 30 μg of total protein was run on a Tris-acetate sodium dodecyl sulfate (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, Hercules, CA, USA) at 130 V in an ice-water bath. Subsequently, the proteins were wet-transferred to a nitrocellulose membrane (Sigma–Aldrich). All of the immunodetection steps were performed using the SNAPid system (Millipore). The primary antibodies anti-huntingtin (1:1000, MAB2166, Millipore) and anti-plectin (1:1000, ab83497, Abcam, Cambridge, UK) and secondary antibodies anti-mouse HRP-conjugate (1:2000, A9917, Sigma–Aldrich) and anti-rabbit HRP-conjugate (1:2000, 711-035-152, Jackson ImmunoResearch) were used in a PBS/0.1% Tween-20 buffer containing 0.25% non-fat milk. The immunoreaction was detected using WesternBright Quantum HRP Substrate (Advansta, Menlo Park, CA, USA). The protein bands were scanned directly from the membrane using a camera and were quantified using Gel-Pro Analyzer.