Western blot analysis

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.