Western blot.

Protein prepared from kidney lysates was immunoblotted following a standard protocol. Briefly, mouse kidney tissues were homogenized in RIPA buffer (150 mM NaCl, 50 mM Tris·HCl, pH 7.4, 5 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, and 1× protease inhibitor cocktail from Sigma-Aldrich), and the extracted proteins were fractionated by SDS-PAGE and subsequently transferred to PVDF membranes for immunodetection with specific primary antibodies and correspondent HRP-conjugated secondary antibodies. Primary antibodies used in this study were anti-FPN (1:1,000, ab78066 and Ab85370, Abcam; 1:1,000, sc-49668, Santa Cruz Biotechnology; 1:1,000, MTP11-S, Alpha Diagnostic International); anti-FTH1 (1:5,000, ab75973, Abcam), anti-NADPH oxidase-4 (NOX4) (1:5,000, A1528, Abclonal), anti-glutathione peroxidase-1 (GPx1) (1:2,000, A0873, Abclonal), anti-GPx4 (1:2,000, A1933, Abclonal), anti-GPx6 (1:1,000, sc292680, Santa Cruz Biotechnology), anti-catalase (1:2,000, ab16731, Abcam), anti-heme oxygenase-1 (HO-1; 1:2,000, A1346, Abclonal), anti-receptor-interacting Ser/Thr kinase-3 (RIPK3) (1:2,000, ab56164, Abcam), anti-p-RIPK3 (1:2,000, phosphorylation at Tyr²³¹ and Ser²³², ab201912, Abcam), anti-Bax (1:1,000, 556467, BD Biosciences), anti-cleaved caspase-3 (1:2,000, 9664, Cell Signaling), and anti-β-actin (1:5,000, HC201, Transgene). All HRP-conjugated secondary antibodies and ECL kits were purchased from Jackson Immunoreseach Laboratories and Thermo Scientific, respectively. After secondary antibodies had been applied, the proteins were visualized by ECL-based chemiluminescence using an ImageQuant LAS 4000 mini system (GE Healthcare). Protein abundance was quantified based on densitometry using Fiji software (Scion, Frederick, MD). Protein abundance was expressed relative to β-actin.