Western Blot Analysis of GSK3β and β-Catenin

Western blot analysis was performed, according to the previous method (Cho et al., 2018 (link); Wu et al., 2015 (link)). The right hemisphere was homogenized on ice, and lysed in a lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, 1.5 mM MgCl₂·6H₂O, 1 mM sodium orthovanadate, and 100 mM sodium fluoride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Protein samples (30 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane. Mouse GSK3β antibody, mouse phosphorylated GSK3β (p-GSK3β) antibody, mouse β-catenin antibody, and mouse phosphorylated β-catenin (p-β-catenin) antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody (1:2,000; Vector Laboratories) for GSK3β, p-GSK3β, β-catenin, and p-β-catenin were used as secondary antibodies. Using a cold pack and prechilled buffer, membrane transfer was conducted at 4°C. Enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) was used for band detection.