Protein Extraction and Western Blot Analysis

Hippocampal or cell proteins were extracted using RIPA lysate (containing protease inhibitors, Solarbio, China). Samples were completely disrupted at 4 °C for 30 min, followed by centrifugation at 13,000 g for 15 min, and the determination of total protein concentration by bicinchoninic acid assay (BCA, Solarbio). After density at 95°C for 10 min, 30 μg of protein was loaded onto an SDS-PAGE gel for subsequent electrophoresis. Western blotting was performed as previously described 11 (link), 41 (link), 42 (link). ChemiDoc XRS (Bio-Rad, USA) was used to visualise immunoreactive proteins. The relative intensity of the bands was quantified using the Image Lab Analyzer software (Bio-Rad). The antibodies used in this study are listed in Table 1.