Western blot analysis

Brain tissues were added to a protein extraction reagent (Beijing Biolab Technology Co., Ltd., Beijing, China) according to a 1: 10 (g/l) ratio. The components of the reagent were 20 mM Tris (pH, 7.5), 150 mM NaCl, 1% Triton X-100, Na2HPO4, β-glycerophosphate, ethylenediamine tetraacetic acid (EDTA), Na3VO4, PVP40 and leupeptin. The extracted protein was centrifuged at 12,500 rpm for 15 min to obtain the supernatant, and the protein was quantitatively determined by the bicinchoninic acid (BCA) kit (Beyotime Institute of Biotechnology, Jiangsu, China). A sample with the same amount of the protein was taken and diluted to 20 µl with 4× loading buffer and normal saline. After denaturation at 100°C for 5 min, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was prepared according to the relative molecular weight of the target protein. The proteins were transferred onto the membrane according to the concentration and loading quantity of the protein. Five percent skim milk was used to block the membrane for 1 h. After gently shaking at 37°C for 2 h, GAPDH (ab9485, 1:5,000) and the primary antibodies BACE1 (ab108394, 1:1,000), PI3K (ab86714, 1:1,000), phosphorylated (p)-PI3K (ab182651, 1:1,000), Akt (ab8805, 1:500), p-Akt (ab8933, 1:1,000), caspase-3 (ab13847, 1:500) and cleaved-caspase-3 (ab49822, 1:500; all purchased from Abcam, Cambridge, MA, USA) were added for incubation at 4°C overnight. The membrane was washed 3 times, each time for 5 min. POD-conjugated goat anti-rabbit antibody (A5795, 1:5,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to incubate for 1.5 h at 37°C, and the membrane was washed 3 times for 10 min each time. Luminescent reagent was added, and the film was exposed by X-ray, developed and fixed in the darkroom. Quantity One software was employed for the semiquantitative analysis for BACE1 expression. The ratio of the gray value of the internal reference to that of BACE1 on film represented the expression level of BACE1. This method was also applied to detect the protein expression of caspase-3 and PI3K/Akt signaling pathway-related genes.