Alexa fluor 680 790 labeled goat anti rabbit goat anti mouse igg antibody

Total protein from the glomeruli and podocytes was extracted with RIPA buffer (Beyotime, China) mixed with a protease inhibitor cocktail (Sigma- Aldrich, USA). The extractions were then centrifuged at 13,000 rpm for 10 min at 4°C. The supernatants were mixed with loading buffer prior to being boiled at 100°C for 5 min. Equal amounts of protein samples were separated through SDS-PAGE and then transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with milk for 1-2 h before incubation with a primary antibody (Sirt6 rabbit monoclonal antibody, 1:1000, Abcam; p-AMPKα1/2 (Thr172) rabbit polyclonal antibody, 1:100, Santa Cruz Biotechnology; AMPKα1/2 (H-300) rabbit polyclonal antibody, 1:100, Santa Cruz Biotechnology; caspase-3 mouse monoclonal antibody, 1:1000, Novus Biologicals; GAPDH rabbit monoclonal antibody, 1: 1,000, Antgene and GAPDH mouse monoclonal antibody, 1: 1,000, Antgene) overnight at 4°C. An Alexa Fluor 680/ 790-labeled goat anti-rabbit/goat anti-mouse IgG antibody (1:10,000, LI-COR Biosciences, USA) was used as the secondary antibody, and the blots were visualized using a LI-COR Odyssey Infrared Imaging System.