Western blotting analysis of LC3, BNIP3 and Beclin-1

Renal tissues samples from different sets of rats were homogenized using RIPA Lysis Buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and phosphatase and protease inhibitors) (Beyotime, Nanjing, China), and the protein lysates were quantified using BCA Protein Assay Kit (Beyotime, Nanjing, China) according to the manufacturer’s protocol. The protein lysates were separated on 12% SDS-PAGE (100 V for 2h) followed by transfer to PVDF membrane (Millipore, Billerica, MA) at 100 mA for 1h at 4°C. The PVDF membrane was then blocked by 5% skimmed milk for 1h followed by incubation with corresponding rabbit anti-rat primary antibodies overnight at 4°C. The antibodies included anti-LC3 (1:1000; 2775S, Cell Signaling, Danvers, MA), anti-BNIP3 (1:1000; 3769S, Cell Signaling, Danvers, MA) and anti-beclin-1 (1:1000; 3495S, Cell Signaling, Danvers, MA). Then, after washing, the membranes were incubated with the goat anti-rabbit fluorescent secondary antibody (1:15000; C30815-02, LICOR, USA) for 1.5h and analyzed by Odyssey infrared fluorescence scanning imaging system (Odyssey, LICOR, USA). GAPDH (1:10000; ab181602, Abcam, Cambridge, MA, USA) was used as internal control.