Anti lc3 β

Cultural NRK-52E cells were lysed in 1 × SDS sample buffer. The kidneys were lysed with RIPA solution containing 1% NP40, 0.1%SDS, 100 mg/ml PMSF, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail (Sigma, St Louis, MO) on ice. The supernatants were collected after centrifugation at 13,000 × g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay. An equal amount of protein was loaded into 10% or 15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The primary antibodies were as follows: anti-LC3-β (cat: L7543, Sigma Aldrich, St Louis, MO), anti-cleaved caspase3 (cat: 9664, Cell Signaling Technology, Beverly, MA), anti-β-actin (cat: sc-1616, Santa Cruz Biotechnology), anti-p-AMPKα (T172) (cat: 2535, Cell Signaling Technology, Beverly, MA), anti-AMPKα (cat: 5831, Cell Signaling Technology, Beverly, MA), anti-p-S6 (S235/236) (cat: 4857, Cell Signaling Technology, Beverly, MA). Quantification was performed by measuring the intensity of the signals with the aid of National Institutes of Health Image software package.

Frozen hippocampi or cultured cells were homogenized in ice-cold RIPA buffer (Beyotime Biotechnology, Jiangsu, China) containing 0.5 mM PMSF. Insoluble material was removed by centrifuging the homogenates at 16 500 g for 15 min at 4 °C. The protein concentrations of the supernatants were quantified using a BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China). Protein samples were boiled for 5 min with loading buffer, and proteins (30 μg per well) were separated by 15% SDS-polyacrylamide gel electrophoresis (Beyotime Biotechnology, Jiangsu, China) and then transferred onto nitrocellulose membranes (Millipore, Watford, UK). Membranes were blocked with 5% nonfat milk in PBST for 1 hour and then incubated at room temperature with PBST solution containing 3% BSA. Subsequently, the following primary antibodies were added: anti-Atg5 (Santa Cruz, CA, USA), anti-Beclin1 (Santa Cruz, CA, USA), anti-LC3β (Sigma-Aldrich St. Louis, USA), anti- p62 (Santa Cruz, CA, USA), anti-β-actin (Santa Cruz, CA, USA), anti-caspase-3,-8,-9 (Cell Signaling Technology, MA, USA), anti-bax (Santa Cruz, CA, USA), and anti-bcl-2 (Santa Cruz, CA, USA). Membranes were washed and incubated with horseradish peroxidase-conjugated anti-IgG antibody at room temperature for 1 hour. Bands were revealed by chemiluminescence, and band intensities were quantified using ImageJ software.