Western Blot Analysis
Hippocampus samples were collected in RIPA buffer (Beyotime, Shanghai, China) and centrifuged for 20 min at 12 000 × g and 4°C. Then we collected supernatants calculated protein concentration. We took 40 to 60μg protein per sample, and performed protein separation by SDS-PAGE electrophoresis. After blocked by the 5% milk powder at room temperature for 2h,the membranes were incubated with primary antibodies against pERK (Cell Signaling Technology, #4370), ERK (Cell Signaling Technology, #4695), OTR(Abcam, #ab217212), nNOS (Cell Signaling Technology, #4231), GAPDH (Cell Signaling Technology, #5174) at 4°C overnight. The membrane was incubated with a secondary antibody at room temperature for 1h. We detected signals by enhanced chemiluminescence following the manufacturer’s recommendations.