Immunoblotting

Half-brain sections from 6-month-old mice were homogenized in a non-denaturing lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM PMSF), supplemented with protease and phosphate inhibitor cocktails (Sigma-Aldrich cat# 4693132001 and 4906837001). The homogenates were cleared by centrifugation at 13,000 × g at 4°C for 25 min, and the protein concentration in the collected supernatant was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein extracts (20 μg of protein for each sample), were incubated in a boiling bath for 10 min and analyzed by SDS-PAGE on 4–20% precast polyacrylamide gradient gel (Bio-Rad, 4561096). Western blot analyses were performed according to standard protocols using antibodies against MBP (1:1000, Abcam, cat# ab218011), MAG (1:1000, Abcam, cat# ab89780), and α-tubulin (1:2000, DSHB, cat# 12G10) as a control. The immunoblots were revealed by chemiluminescence with SuperSignalWest Pico PLUS (Thermo Fisher Scientific, Waltham, MA, USA). Detected bands were quantified using ImageJ 1.50i software (National Institutes of Health, Bethesda, MD, USA) and normalized for the intensity of the α-tubulin band.