2.9. Western blot analysis

SH-SY5Y cells or brain tissues were both extracted using RIPA buffer. The rat cortex and hippocampus were carefully and rapidly separated from brains on ice. Each 0.1 g tissue was added with 1 mL RIPA buffer and homogenized. The homogenate was then lysed on ice for 30 min. A BCA protein assay kit was used for the protein quantification. Proteins were loaded and separated using SDS-PAGE method, then transferred onto a PVDF membrane. After being blocked with 5% BSA for 2 h, proteins were treated with primary antibodies against p-FOXO3a, FOXO3a, p-AKT, AKT, BIM, cleaved caspase 3 and GAPDH at 4 °C overnight. Then the secondary antibodies against HRP-conjugated rabbit or mouse IgG were added to the system. An ECL method was used for image developing. Band density was calculated with the ImageJ software.