Acsl4

Total protein was extracted from rat ileums, and the concentration of total protein was determined by BCA Protein Assay kit (TransGen, Beijing, China). Western blotting was performed with the following primary antibodies: ACSL4 (Abcam), GPX4 (Abcam), FTH1 (Abcam), ZO-1 (Abcam), occludin (Zymed Laboratories), claudin-1 (Santa Cruz Biotechnology), and claudin-2 (Zymed Laboratories). β-Actin (Sigma) was used as loading control. For protein quantification, the density of Western blot bands was measured by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).

Tissue lysates were homogenized in SDS lysis buffer [Cell Signaling Technology (CST), #7722] and transferred onto polyvinylidene difluoride membranes. After blocking in 5% milk in tris-buffered saline and 0.1% Tween 20, membranes were incubated overnight with the following antibodies at 4°C: CASP9 (Abcam, #ab202068), cleaved CASP3 (CST, #9664), RIP3 (Millipore Sigma, #PRS2283), BAX (CST, #2772), BCL2 (CST, #3498), GSDMD (Santa Cruz Biotechnology, sc-393656), NLRP3 (CST, #15101), ACSL4 (Abcam, #ab155282), LC-3 (CST, #2775S), cGAS (CST, #31659), STING (CST, #13647S), TBK1 (CST, #38066), pTBK1 (CST, #5483), IRF3 (CST, #4302), pIRF3 (CST, #4947), p65 (CST, #8242), pp65 (CST, #3033), and GAPDH (CST, #5174). Membranes were incubated with horseradish peroxidase–conjugated secondary anti-rabbit or anti-mouse antibody (CST, #7074 and #7076) for 1 hour at room temperature. Signal was detected by enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, #34094; Thermo Fisher Scientific). Western blots were quantified using the Fiji software (58 (link)).

Frozen heart tissues were lysed mechanically in cold RIPA buffer. Proteins were extracted and quantified using a BCA protein assay kit (Best Bio, Shanghai, China). The proteins were electrophoretically separated using SDS-PAGE and transferred to a polyvinylidene difluoride membranes (Millipore, United States). The membranes were blocked with 3% BSA for 1.5 h at room temperature, and antigens were detected using the following antibodies at 4°C overnight, CSE (1:1,000, Proteintech, United States), CBS (1:1,000, Proteintech, United States), 3-MST (1:5,000,SantaCruz, United States), P53 (1:2000, Proteintech, United States), P21 (1:2000, Proteintech, United States), NRF2 (1:2000, Proteintech, United States), SLC7A11 (1:2000, Proteintech, United States), ACSL4 (1:2000, Abcam, United States), GPX4 (1:2000, Proteintech, United States), FPN1 (1:1,000, Proteintech, United States), FTH (1:1,000, Immunoway, United States), TRF1 (1:1,000, Abcam, United States), and GAPDH (1:5,000, Proteintech, United States). The blots were incubated with a horseradish peroxidase-conjugated anti-rabbit (Proteintech, United States) or anti-mouse (Proteintech, United States) secondary antibody at room temperature for 1.5 h. All antibodies were diluted with TBST. Blot bands were visualized by enhanced chemiluminescence and detected by ImageJ software (1.52, NIH, United States).

Total protein from pancreatic tissue was obtained by RIPA lysis buffer with 1% phenylmethylsulfonylfluoride (Solarbio). The proteins were electrophoresed on 10% and 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). After being blocked in 5% skimmed milk for 2 h at 25 °C, the membranes were cut prior to hybridisation with primary antibodies. After that, the membranes were incubated at 4 °C overnight with primary antibodies: GPX4 (1:1000; BOSTER), xCT (1:1000; Abcam), ACSL4 (1:1000; Abcam), LPCAT3 (1:500; ABclonal, Wuhan, China), binding immunoglobulin protein (Bip, 1:1000; Zenbio, Chengdu, China), C-EBP homologous protein (CHOP, 1:1000; BOSTER, Wuhan, China), eukaryotic initiation factor 2α (EIF2α, 1:2000; Proteintech, Wuhan, China), phosphoration of EIF2α (p-EIF2α, 1:1000; Abcam), and β-actin (1:1000; Cell Signaling Technology, Danvers, MA, USA). Subsequently, the membranes were incubated with a secondary goat anti-rabbit antibody (1:10,000; Cell Signaling Technology) for 2 h at 25 °C. Protein bands were visualized using the Odyssey Fc Imaging System, and band images were analyzed using Image J software. All initial western blotting bands in this study are presented in the Supplementary Information.

The proteins were isolated and separated by SDS‐PAGE. Primary antibodies against CYGB (#60228, Proteintech), YAP1 (#14074, Cell Signaling Technology), phospho‐YAP1 (Ser127) (#13008, Cell Signaling Technology), p53 (#2524, Cell Signaling Technology), phospho‐p53 (Ser15) (#9284, Cell Signaling Technology), SLC7A11 (#12691, Cell Signaling Technology), ACSL4 (#ab155282, Abcam) and GAPDH (#AP0063, Bioworld Antibodies) were used.

Protein sample tissues were collected from the peri-infarct cortex. Proteins were separated on SDS/PAGE gels (10%) and electroblotted into a PVDF membrane. The PVDF membranes were then incubated in 5% nonfat milk at room temperature for 1 h and then incubated with primary antibodies overnight at 4°C. The membrane was washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween-20 and incubated in the secondary antibodies (ABclonal, China) for 1 h at room temperature. We used the Bio-Rad system to scan the proteins (n = 3/group). The primary antibodies used in this experiment were listed as follows: GAPDH (Proteintech, China), GPX4 (Abmart, China), HIF-1α (Zen-Bio, China), and ACSL4 (Abcam, UK).