Western Blotting of Insulin Signaling Pathway

Western blotting was performed, as the previously described method (Kim et al., 2020 (link)). Hippocampal tissues were homogenized on ice and lysed in lysis buffer. The protein content was measured using a colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Thirty micrograms of protein were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred on to a nitrocellulose membrane. Then, incubated with mouse β-actin (1:1,000; Santa Cruz Biotechnology), total IR β (t-IRβ) and phosphorylated IRβ (p-IRβ) (1:1,000; Cell Signaling Technology, Danvers, MS, USA), total IR substrate 1 (t-IRS-1) and phosphorylated IRS-1 (p-IRS-1) (1:1,000; Cell Signaling Technology), t-PI3K and p-PI3K (1:1,000; Cell Signaling Technology), total 3-phosphoinositide dependent protein kinase-1 (t-PDK1) and phosphorylated PDK1 (p-PDK1) (1:1,000; Cell Signaling Technology), total-Akt (t-Akt) and phosphorylated Akt (p-Akt) (1:1,000; Cell Signaling Technology), total glycogen synthase kinase 3 beta (t-GSK3β) and phosphorylated GSK3β (p-GSK3β) (1:1,000; Cell Signaling Technology). Horseradish peroxidase-conjugated secondary anti-mouse antibodies were used for β-actin and horseradish peroxidase-conjugated secondary anti-rabbit antibodies were used for t-IRβ, p-IRβ, t-IRS-1, p-IRS-1, t-PDK, p-PDK, t-PI3K, p-PI3K, t-Akt, p-Akt, t-GSK3β, and p-GSK3β.