Bnip3l nix

The renal cortex was homogenized with RIPA Lysis Buffer. After centrifugation, protein concentration was determined by bicinchoninic acid (BCA) protein assay kit. The protein samples were then denatured in boiling water for 10 min. Equal amounts of proteins were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin in tris-buffered saline (TBS) for 1 h and incubated overnight at 4°C with following antibodies: Parkin (Cell Signaling Technology, cat. no. 2132), DRP1 (Abcam, cat. no. ab184247), LC3B (Cell Signaling Technology, cat. no. 2775), BNIP3L/Nix (Cell Signaling Technology, cat. no. 12396), caspase3 (Cell Signaling Technology, cat. no. 9662), cleaved caspase3 (Cell Signaling Technology, cat. no. 9661), and β-actin (Sigma, cat. no. A1978). Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, followed by enhanced chemiluminescence reaction. Quantification was performed by the Gel and Graph Digitizing System. The full western blot bands of LC3B, Parkin, BNIP3L, DRP1, caspase3, cleaved caspase3 were shown in Supplementary Figures S1–S6.

Renal tissues and cells were homogenized in RIPA buffer containing Protease/Phosphatase Inhibitor Cocktail (Beijing Suo Lai Bao Technology Co., Ltd.) after washing with PBS, and total protein concentration was estimated using BCA Protein Assay Kit (Wuhan Doctorate Bioengineering Co., Ltd.). Protein samples (40-80μg) were submitted to SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were blocked for 2 h in 5% non-fat milk and then incubated with primary antibodies against LC3 (1:1,000, Cell Signaling), SQSTM1/p62 (1:1,000, Abcam), BNIP3L/Nix (1:1,000, Cell Signaling), mTOR (1:1,000, Cell Signaling), Phospho-mTOR (1:1,000, Cell Signaling), AMPKα (1:1,000, Cell Signaling), Phospho-AMPKα (1:1,000, Cell Signaling), SGLT2 (1:1,000, Abcam), and β-actin (1:500, Sanjian Biotechnology) at 4°C overnight. After being washed with TBST, the membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000, Ai Meijie Technology Co., Ltd, China). The reactive bands were detected using the ECL system (Advansta). Signal intensity was then assessed using automatic gel imaging system (SYNGENE). Studies were replicated 3 times.

Antibodies targeting the following proteins were used: TFRC (ab214039, Abcam, Cambridge, UK), FPN (NBP1-21502, Novus Biologicals, Englewood, CO, USA), FTH1 (ab65080, Abcam), SLC7A11 (600-401-GU3, Rockland Immunochemicals, Pottstown, PA, USA), GPX4 (ab125066, Abcam), GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), PGC1α (NBP1-04676, Novus Biologicals), SHARPIN (ABF128, Millipore, Burlington, MA, USA), β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), VDAC1/3 (ab14734, Abcam), PRMT5 (sc-376937, Santa Cruz Biotechnology), SDMA (SYM10; 07-412, Millipore), SOX10 (sc-365692, Santa Cruz Biotechnology), MITF (ab12039, Abcam), LC3B (NB100-2220, Novus Biologicals), NRF2 (#12721, Cell Signaling Technology), Parkin (#4211, Cell Signaling Technology), and BNIP3L/NIX (#12396, Cell Signaling Technology). For NRF2 detection, samples were pretreated with 50 μM MG132 (FUJIFILM Wako Pure Chemical Corporation, San Diego, CA, USA) for 3 h. Complex I/III/V was detected using Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam). Erastin, EPZ015666, ATN-161, and RGD peptide were purchased from Selleck Chemicals (Houston, TX, USA). TGF-β was purchased from BioLegend (San Diego, CA, USA). RSL3, SC-514, and ferrostatin-1 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Deferoxamine was obtained from Cayman Chemicals (Ann Arbor, MI, USA).