Af6348

The hippocampus was carefully removed before extracting the total protein on ice. After adding 360 μL RIPA and 3.6 μL PMSP solution (both from Biyuntian Technologic Inc., China), the homogenate was centrifuged at 12,000 rpm for 10 min. The supernatant was collected and the concentration of protein was determined using a BCA protein assay kit (Biyuntian Technologic Inc.). Equal amounts of protein were separated by SDS-polyacrylamide gels and then transferred onto PVDF membranes (Solarbio Science & Technology Co., Ltd., China). The membranes containing P-Akt were blocked in 5% bovine serum albumin (BSA), whereas the others were blocked with 5% non-fat milk for 2 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: anti-p-Akt (1:1000, #AF0016; Affinity Bioscience, China), anti-Akt (1:1000, #AF6261; Affinity Bioscience), anti-caspase-9 (1:1000, #AF6348; Affinity Bioscience) and anti-β-actin (1:1000, #TA-09;Zhongshan Biotech, China). The membranes were washed with TBST (4 × 8 min) and incubated for 2 h with HRP-conjugated secondary antibodies (#ZB2301; Zhongshan Biotech): goat anti-mouse IgG (1:5000) and goat anti-rabbit IgG (1:8000). The blots were developed using an enhanced chemiluminescence kit and exposed to X-ray film. The protein expression levels were analyzed using Image J software (NIH, Bethesda, MD, USA).