Anti gpx4

Protein lysates were obtained using RIPA buffer supplemented with protease and phosphatase inhibitors. The protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (#ZJ101, Epizyme, China). Western blot analysis was conducted following previously described methods (Chen et al., 2020 (link)). The study utilized several primary antibodies, including anti-NCOA4 (#A5695, 1:1,000), anti-GPX4 (#A21440, 1:1,000), anti-PCBP1 (#A1044, 1:1,000), anti-collagen II (#A19308, 1:1,000), anti-p16 (#A0262, 1:1,000), and anti-β-actin (#AC026, 1:1,000), all of which were purchased from ABclonal.

Western blot was performed according to the standard protocol described in a previous study (Xie et al., 2019 (link)). The collected ipsilateral cerebral cortical samples were fully lysed using protease inhibitors (Beyotime, China) and RIPA lysis buffer (Thermo Fisher, United States). An equal amount of protein was separated in each lane by performing SDS-PAGE, and the protein was transferred onto PVDF membrane (Millipore, United States). The membrane was blocked by using 5% nonfat milk for 1 h at RT, and then was incubated with primary antibody overnight at 4°C. The membrane was washed with TBST for three times, each of which lasted 5 min. Afterwards, secondary antibody was added for 2 h of incubation at RT. The intensity of signal was detected by chemiluminescence using ECL substrate (Vazyme, China). ImageJ software was used for the comparison of signal intensity. The following primary antibodies were used: anti-SLC7A11 (1:500, cat#A15604; Abclonal); anti-GPX4, (1:500, cat# A1933, Abclonal); Anti-Nrf2 (1:1000, cat# A11159, Abclonal); Anti-Histone H3 (H3), (1:5000, cat# AF0009, Beyotime); Anti-HO-1 (1:1000, cat# ab13248, Abcam); Anti-Tubulin, (1:2000, cat# 2148S, Cell signaling technologies); Anti-GAPDH (1:5000, cat# 5174S, Cell signaling technologies).

Renal tissues and HK-2 cells were lysed using radioimmunoprecipitation assay buffer (Servicebio). The lysates were subjected to polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membrane was occluded with 5% skim milk to prevent nonspecific binding, followed by overnight incubation with primary antibodies. Next, the membrane was washed with TBST and incubated with HRP-conjugated secondary antibodies for 1 h. Immunoreactive signals were formulated using an enhanced chemiluminescence kit (Biosharp, China) and visualized using a ChemiDoc MP system. We used ImageJ to calculate the relative protein density. Every experiment was conducted with three replicates. The following are the primary antibodies used in the analysis: anti-EZH2 (5246S; 98 kDa; 1:1000; CST), anti-H3K27me3 (9733S; 17 kDa; 1:1000; CST), anti-H3 (17,168–1-AP; 17 kDa; 1:2000; Proteintech), anti-SOX4 (A10717; 47 kDa; 1:1000; Abclonal), anti-SLC7A11 (A2413; 55 kDa; 1:1000; Abclonal), anti-GPX4 (A11243; 19 kDa; 1:1000; Abclonal), anti-acyl-CoA synthetase long-chain family 4 (ACSL4) (A6826; 79 kDa; 1:1000; Abclonal), anti-prostaglandin-endoperoxide synthase 2 (PTGS2) (66,351–1-lg; 68 kDa; 1:1000; Proteintech), and anti-GAPDH (10,494–1-AP; 37 kDa; 1:5000; Proteintech) antibodies.