Anti slc7a11

Approximately 1 million cells for each condition were harvested and washed twice with PBS before resuspension in PBS. FC block (Biolegend, 422301, 1:200) was then added for 20 min at room temperature before the addition of primary antibody at a 1:100 dilution before incubation at 4 °C for 30 min. Cells were then washed 3 times with PBS before resuspension in PBS. Samples were then stored either on ice or at 4 °C protected from light until analysis by flow cytometry using LSR Fortessa (BD Biosciences) instruments.
The following antibodies were used: anti-human CD45-FITC (BioLegend, 304005), anti-mouse CD45-APC (BioLegend, 157605), anti-SLC7a11 (SantaCruz Biotech, SC98552). Downstream analysis was performed using FlowJo version 10.6.1 (BD Biosciences). Example FACS gating strategies are available in Supplementary Information.

Samples from tissues or cells were lysed in RIPA buffer supplemented with 1x protease and phosphatase inhibitor cocktail (Thermo Fisher, America) and incubated on ice for 30 min. After centrifugation at 14000 × g for 10 min at 4 °C, the supernatant was collected to determine the protein concentration by BCA assay. The samples were prepared for electrophoresis, followed by transfer onto PVDF membranes. The primary antibodies used in this work were as follows: anti-ZO-1 (Santa Cruz Biotechnology), anti-p-MLKL (Cell Signaling Technology), anti-p-RIP1 (Cell Signaling Technology), anti-cleaved/full-length caspase 9 (Cell Signaling Technology), anti-cleaved/full-length caspase 3 (Cell Signaling Technology), anti-GPX4 (Santa Cruz Biotechnology), anti-SLC3A2 (Santa Cruz Biotechnology), anti-SLC7A11 (Santa Cruz Biotechnology), anti-ANXA2 (ABclonal), anti-His-tag (ABclonal), anti-HA-tag (ABclonal), anti-HSPB1 (Cell Signaling Technology), and anti-PRDX1 (Cell Signaling Technology). Secondary antibodies were purchased from ABclonal. The membranes were visualized via the chemiluminescence method.

Sigma-Aldrich supplied the folic acid. Ruscogenin was purchased from MedChem Express. CRE, BUN, MDA, GSH, albumin, and carbonylated protein kits were got from the Nanjing Jiancheng Institute of Biological Engineering. NAG activity kits were purchased from Beijing Solaibao Technology Co. Anti-Slc7a11 (Santa Cruz, Santa Cruz, CA, USA), anti-HO-1 (Santa Cruz, Santa Cruz, CA, USA), anti-Rev-erbα (Sigma-Aldrich, St. Louis, MO, USA), anti-Rev-erbβ (Abnova, Taipei, Taiwan, China), anti-GPX-4 (Santa Cruz, Santa Cruz, CA, USA), and anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA) were used in the study. C57BL-6 mice were purchased from Nanjing Qinglong Mountain Animal Breeding Farm.