Hippocampus Protein Analysis by Western Blot

Hippocampus preparation and Western blot were performed as described previously [13 (link), 20 (link)]. Briefly, each hippocampus was dissected immediately after sacrificing the animal and was stored at − 80 °C until use. For immunoblot analysis, the frozen hippocampi were homogenized in ice-cold 50 mM Tris-HCl buffer (pH 7.4) containing 1% Triton X-100, 0.2 mM PMSF, and 1 mM EDTA. Homogenates were centrifuged at 12,000×g for 10 min at 4 °C. The supernatants obtained were immediately placed in boiling water for 10 min. Samples (10 μg protein/lane) were separated on 10% SDS-PAGE gels and electrophoretically transferred to nitrocellulose membranes. The membranes were probed with rabbit antibodies against the following proteins: Tau (C-17) (polyclonal, 1:1000, Bioworld Technology, China), PS202 (polyclonal, 1:1000, Bioworld Technology, China), PS404 (polyclonal, 1:1000, Bioworld Technology, China), Egr1 (polyclonal, 1:1000, Bioworld Technology, China), and c-Fos (polyclonal, 1:1000, Bioworld Technology, China). Anti-GAPDH (1:10,000; Bioworld Technology) was used as the internal reference standard.