Western blot analysis

The expression of GRP78, CHOP or cleaved caspase-12 was measured by Western blot. After sacrifice, the entire hippocampus was removed and homogenized in ice-cold homogenizing buffer (20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF. After centrifugation at 12,000 for 30 min at 4°C, the supernatant was collected and the protein content was subsequently assayed by using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal quantities of total protein (30 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to nitrocellulose membranes. The membranes were incubated in5% milk at room temperature for 2 h. Blots were then incubated with primary antibodies including rabbit monoclonal antibody for CHOP (diluted 1:1000), GRP78 (diluted 1:2000), cleaved Caspase12 (diluted 1:1000), or β-actin (1:2000). After washing with buffer, the blots were incubated in anti-rabbit secondary antibody-conjugated with horseradish peroxide (1:5000) in TBS-T with 5% milk at 4°C overnight. The signal of the immunoblots was visualized using an image analysis system equipped with a software BIO-ID (Vilber Lourmat, France).