Kif3b

Proteins in the hippocampus and cortex (five mice per group) were lysed using radioimmunoprecipitation assay (RIPA, Beyotime) lysis buffer and phenylmethanesulfonyl fluoride (PMSF, Beyotime) (RIPA:PMSF = 100:1) supplemented with protease inhibitors (Roche). After determination of protein concentration using BCA Protein Assay Kit (Beyotime), equal amounts of protein (50 μg) was separated by SDS-PAGE and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MS, USA). Then the PVDF membranes were blocked using 5% nonfat milk for 2 h, incubated with primary antibodies (4 °C) overnight, and incubated with HRP-conjugated secondary anti-rabbit or anti-mouse antibodies (1:5000, Multi Sciences, China) for 1 h at 37 °C. Immunoreactive bands were observed by enhanced chemiluminescent substrate (ECL, Pierce) exposure to X-ray films. At last, the bands were quantified using Image J, and the relative intensity of each band was normalized to the band of β-actin.
Target proteins were detected using the following primary antibodies: Bax (Abcam, 1:1000), Bcl-2 (Abcam, 1:500), LC3 (Sigma-Aldrich, 1:1000), P62 (Cell Signaling Technology, 1:1000), DIC (Millipore, 1:1000), KIF3B (Cell Signaling Technology, 1:1000), and β-actin (Beijing 4A Biotech Co., Ltd, 1:5000).