Ab125066

Cells were washed with PBS, and total protein lysates were collected using a total protein extraction kit and then quantified with the BCA protein assay reagent (KenGen Biotech Co. Ltd., Nanjing, China). Total protein samples (20 μg/lane) were separated by 10%‐15% SDS‐PAGE (Bio‐Rad, USA) and transferred onto a polyvinylidene difluoride (pore size 0.25 μmol/L) membrane at a constant current of 260 mA. After blocking with 5% bovine serum albumin (BSA) for 1 hour, the membranes were incubated with specific primary antibody transferrin receptor (TFR; ab214039, Abcam; ACSL4, ab155282, Abcam; xCT, ab175186, Abcam; GPX4, ab125066, Abcam; ALR, PA5‐48256, Thermo Fisher) at 4°C overnight or 2 hours at room temperature. After washing with Tris‐buffered saline Tween 20 (TBST) three times, all membranes were incubated with peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. Finally, the membranes were washed three times and visualized using the Chemi Doc Imaging System (Bio‐Rad, USA).

Cells were lysed in RIPA buffer that contained a protease/phosphatase inhibitor cocktail (Sigma-Aldrich) at 4 °C for 30 min and used for immunoblotting. The cytosolic fraction for cathepsin B detection was prepared as previously described^{45 (link)}. A total of 20 μg of protein was loaded per lane of an SDS-PAGE gel and separated by electrophoresis. Proteins were transferred onto nitrocellulose (GE Healthcare, IL, USA) or PVDF membranes (IPVH00010, Millipore, MA, USA). Membranes were subsequently probed with primary antibodies and incubated with either goat anti-mouse IgG (Cell Signalling, 7076, MA, USA) or goat anti-rabbit IgG (Cell Signaling,7074) secondary antibodies conjugated with horseradish peroxidase (HRP). Chemiluminescence was detected using an enhanced chemiluminescence (ECL) system (Translab). The following primary antibodies were used: GPX4 (ab125066, Abcam), SCL7A11 (Cell Signalling, 12,691), Nrf2 (ab62352, Abcam), Keap1 (Cell Signalling, 8047), HO-1 (Cell Signalling, 5853), p62 (ab56416, Abcam), LC3 (PM036, MBL), cathepsin B (Cell Signalling, 31,718) and β-actin (Santa Cruz Biotechnology, sc-47778). The result of gels images was cropped and full-length gels and blots are included in the Supplementary Data.

Levels of SIRT2, FPN1, GPX4 and ACSL4 proteins were detected by western blotting. Protein lysates were prepared from the spinal cord (L4-6). Subsequently, 48 μg protein samples were separated using a 12% gradient sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene difluoride (pore size of 0.45 μm) membrane (Bio-Rad). The membranes were blocked in non-fatty dry milk for 2 h at 37°C and then incubated with primary antibodies, including anti-SIRT2 (1:500, ab51023, Abcam), anti-FPN1 (1:1,000, NBP1-21502, Novus), anti-NRF2 (1:1,000, ab137550, Abcam), anti-GPX4 (1:5,000, ab125066, Abcam), anti-ACSL4 (1:10,000, ab155282, Abcam), and anti-GAPDH (1:10,000, 60004-1-Ig, Proteintech) antibodies at 4°C overnight, and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. We analyzed the density of protein bands using the NIHA ImageJ software.

The kidney tissues were fixed in 4% paraformaldehyde solution, and embedded in paraffin. Then, the sections of paraffin-embedded kidney tissues were stained with periodic-acid-Schiff (PAS) and hematoxylin and eosin (H&E). A Digital Slide Scanner (PANNORAMIC SCAN II, 3D HISTECH) was used to scan the stained slides. The inflammatory score and fungal burden of the kidneys were assessed as previously reported [38 (link)–40 (link)].
For immunohistochemistry (IHC) assay, the renal tissues sections were stained with the indicated primary antibodies. The stained sections were scanned by a Digital Slide Scanner. The ImageJ software was used to quantify the proportion of positive cells. The primary antibodies used were as follows: SLC7A11 (xCT) (Abcam, ab307601, 1:500), GPX4 (Abcam, ab125066, 1:100), Ly-6G (Abcam, ab238132, 1:2000), F4/80 (Cell Signaling Technology, 70076T, 1:250) and CD11c (Abcam, ab219799, 1:100).

Cetrimide agar (MilliporeSigma, 22470), lysogeny broth (LB) (MilliporeSigma, L3152), baicalein (MilliporeSigma, 465119), ferrostatin-1 (MilliporeSigma, SML0583), NADPH (MilliporeSigma), glutathione peroxidase (MilliporeSigma G6137), cumene hydroperoxide (MilliporeSigma C0524), Thiol Fluorescent Probe IV (MilliporeSigma 595504), Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225), anti-GPx4 (rabbit monoclonal, Abcam, ab125066), anti–15-LOX (rabbit polyclonal, Abcam, ab23691), anti-actin (mouse monoclonal, MilliporeSigma, A3854, clone AC-15), and secondary antibody, goat anti-rabbit (MilliporeSigma, A0545) were used. pLoxA antibody was purified from rabbit whole blood by Pocono Rabbit Farm and Laboratory as described previously (12 (link), 27 (link)).

Western blotting was performed according to a previously described procedure [26 (link)]. The following antibodies were used to test the expression of protein: anti-BMP4 (EPR6211) (ab124714, Abcam), anti-BMP4 (ab39973, Abcam), and anti-glutathione peroxidase 4 (anti-GPX4) (ab125066, Abcam). Anti-GAPDH (#5174, Cell Signaling Technology) was used as an internal loading control. Experiments were repeated more than three times, and representative images are presented in the article at the end.