Western Blotting Procedure for TDP-43 Detection

Western blotting was carried out as previously described (Phan et al., 2020 (link)). Protein lysates (10 μg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris–HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4–20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 volts for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed overnight at 4°C with TDP-43 antibody (Proteintech, 10,782-2-AP, 1:2,000) and β-actin (Abcam, ab6276, 1:10,000). They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad).