The harvested hippocampal tissues were subjected to expression analysis by Western blotting as described previously [37 (link)38 (link)]. Briefly, the tissues were homogenized in lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40) with protease inhibitors (aprotinin, pepstatin A, and leupeptin) at mg/mL concentrations for protein extraction. The cell lysate was centrifuged (12,000 g for 10 min at 4℃), the supernatant was removed, and the total protein concentration was determined using a protein assay kit (Bio-Rad). Equal amounts of sample protein (50 µg) were electrophoresed in NuPAGE Novex Bis-Tris gradient gels (Invitrogen). The separated bands were then blotted onto nitrocellulose membranes and incubated with blocking solution (0.1% TBST and 5% non-fat milk) for 2 h, followed by incubation with primary antibodies overnight at 4℃. The membranes were washed three times in TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 60 min. Following five to six washes in TBST, immunoreactive bands were visualized using an ECL detection kit (GE Healthcare, Fairfield, CT, USA) and analyzed using Image J software (NIH Image, Bethesda, MD, USA). Protein expression was normalized relative to the expression of β-actin as an internal standard.