Synaptophysin

Brain tissue was rapidly dissected out from hippocampus and temporal cortex and flash frozen in liquid nitrogen. Tissue samples were homogenized in RIPA buffer with protease and phosphatase inhibitors (Roche). Samples were centrifuged, and protein was collected from supernatant. Primary antibodies were PSD-95 (ThermoFisher, MA1-045) at 1:1000, synaptophysin (Abcam, ab32127) at 1:1000, beta-actin (mAbcam 8226) at 1:5000. Secondary antibodies were HRP-tagged anti-mouse and anti-rabbit (Santa Cruz) 1:5000. Group size was 3.

Four mice per group were anesthetized with intraperitoneal injections of zoletil (90 mg/kg) and xylazine (10 mg/kg), and their hippocampi were collected. The extracted proteins were quantified by the BCA Protein Assay System (Pierce, USA). The proteins were subjected to SDS-PAGE, followed by electro-transfer to PVDF membranes (Millipore, USA). After blocking with 5% skim milk in 0.1% Tris-buffered saline (TBS)/Tween-20, the blots were probed with primary antibodies against GluA1 (1:1000, Abcam), GluN1 (1:1000, Sigma-Aldrich), GluN2A (1:2000, Millipore), NR2B (1:500, BD Transduction Laboratories), and synaptophysin (1:4000, Abcam) overnight at 4 °C. After incubation, the membranes were subsequently incubated for 2 h at room temperature with the appropriate secondary antibodies conjugated with horseradish peroxidase (Vector, USA). The proteins were visualized with an enhanced chemiluminescence kit (Pierce, USA) and band intensities were quantified using the Kodak Gel Logic 2200 imaging system with Molecular Image analysis software (Kodak, USA). β-Actin was used as a loading control (1:10000, Sigma-Aldrich). Data are presented as an average of at least three experiments and analyzed for statistical significance by using Mann-Whitney tests (Prism; Graph Pad, USA).