Slc7a11

Western blot analysis was performed as previously described [16 (link)]. Briefly, the collected cells were lysed with RIPA to extract protein. Afterwards, the samples were transferred to nitrocellulose filter membranes and 5% skim milk powder solution, and then, the primary antibody (GPX4, SLC7A11, NQO1, HO-1, 1:1,000 dilution, Abcam, Cambridge, USA; NRF2, 1:1,000 dilution, Beyotime, China) was incubated at 4°C overnight. The membranes were then washed and incubated with the secondary antibody. β-Actin (1:2,000 dilution, CST, Danvers, USA) was used as an internal control.

The sample was added to a 10% gel for electrophoresis. After electrophoresis, the protein was transferred to the membrane (Millipore, #IPVH00010) and then hybridized with the antibody. Finally, the signal on the membrane was detected by an ECL kit (Thermo, #34096). Antibodies: GPX4 (Abcam, #ab125066), SLC7A11 (Abcam, #ab175186), ACSL4 (Abcam, #ab155282), NRF2 (Proteintech, #16396-1-AP), FSP1 (Proteintech, #20886-1-AP), β-actin (CST, #4970s), PI3K (CST,#4249s), AKT (CST,#9272s), P-AKT (CST,#4060s), and mTOR (CST,#2983s).

Cells and renal tissue were lysed in cold RIPA buffer containing protease inhibitor cocktails. The total protein concentrations were quantified using a BCA protein assay kit. Equal amounts of total protein were separated in 10–15% SDS-PAGE, and samples were transferred onto nitrocellulose membranes, blocked with 5% skimmed milk in TBS/0.5% Tween (TBST) for 1 hour, washed with TBST for three times, and incubated with primary antibodies against MFN2 (1 : 500, Affinity Biosciences), UCP2 (1 : 500, Affinity Biosciences), NRF2 (1 : 500, Proteintech), Slc7a11 (1 : 1000, Abcam), GPX4 (1 : 500, Proteintech), and β-actin (1 : 1000, Proteintech) overnight at 4°C. The nitrocellulose membranes were washed with TBST for three times, 10 min each, and then incubated with secondary antibodies (1 : 10000, Proteintech) for 1 h at room temperature and then washed with TBST for three times again and added with the enhanced ECL Chemiluminescent Substrate Kit (Yeasen Biotechnology, Shanghai, China) in the membrane. The relative intensity of the target band was detected using the AmerSham Imager 600 image system (GE, USA). Images were quantified using ImageJ software (NIH, USA).

Western blotting and quantitative PCR (RT‐qPCR) were performed following the previous report.
⁴³ (link) Following antibodies were used in Western blotting: Actin, FSP1, ACSL4, SLC7A11, VDAC2, and GPX4 rabbit polyclonal antibodies (1:1000; Abcam); VCAM‐1 and ICAM‐1 (1:500; Santa Cruz Biotechnology, Inc, Dallas, TX, USA). The primers used in RT‐qPCR are shown in Table S1.

Total protein samples were extracted and the concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Sample lysates (10 μg of protein) were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was incubated with specific antibodies for SLC7A11 (1:1,000) or cyclin D1 (1:3,000; Abcam, Cambridge, MA, USA) at 4°C overnight followed by incubation with secondary antibodies. Protein levels were normalized to those of total GAPDH using a monoclonal anti-GAPDH antibody (Sigma-Aldrich Corporation, St. Louis, MO, USA). Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad).

Standard western blot analysis was performed with antibodies for DNMT3A (Cell Signaling Technology, Danvers, MA, USA), SLC7A11 (Abcam, USA) and β-actin (Santa Cruz, CA). The protein bands were detected by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL).