A16848

Western blotting was performed following standard procedures. Anti-ACSL4 antibody (1:1,000, A16848, ABclonal, Woburn, MA, USA), anti-ferritin Heavy Chain (FTH) antibody (1:1,000; 381204, Zen Bio), anti-GPX4 antibody (1:5,000; ab125066, Abcam, Cambridge, UK), anti-ERK1/2 antibody (1:2,000; YT1625, Immunoway, Plano, TX, USA), anti-Phospho-Erk1/2 antibody (1:2,000; O1923, Cell Signaling, Danvers, MA, USA), anti-MEK1/2 antibody (1:2,000; D1A5, Cell Signaling, Danvers, MA, USA), anti-Phospho-MEK1/2 antibody (1:2,000; 41G9, Cell Signaling, Danvers, MA, USA) and secondary antibody (1:2,000; Beyotime, Jiangsu, China) were used to detect protein expression. Anti-GAPDH (Zen Bio) was used at a 1:2,000 ratio. Images were acquired by a Tanon 5500 imaging system (Tanon, Shanghai, China). The images were scanned with the Image J scanning software, and the data was expressed as relative values to sham or control values.

The liver tissue was homogenized in lysis buffer. After centrifugation, the protein concentration of the lysate was measured using BCA Protein Assay Kit (Beyotime, P0012, China), in line with the manufacturer's protocols. Equivalent amounts (50 μg) of protein were separated on SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked with 5% skimmed milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated with antibodies against COX2 (1 : 1000, 12375-1-AP, Proteintech), ACSL4 (1 : 1000, A16848, ABclonal), GPX4 (1 : 1000, BM5231, Boster), FTH1 (1 : 1000, A19544, ABclonal), Nrf2 (1 : 1000, 16396-1-AP, Proteintech), HO-1 (1 : 1000, 10701-1-AP, Proteintech), or GAPDH (1 : 10 000, 60004-1-AP, Proteintech) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies against rabbit or mouse IgG (1 : 5000, LF101 and LF102, respectively, Epizyme) for 1 hour at room temperature. A Bio-Rad immunoblot analysis detection system (Bio-Rad, Hercules, CA, USA) was used for visualization. Protein band densities were assessed by ImageJ analysis software, and the relative densities against the loading control (GAPDH) were calculated.

The hippocampal tissues, primary cultured neurons, and cell lines were homogenized using RIPA buffer (Beyotime, P0013B) with a 1 × protease inhibitor cocktail (Beyotime, P1010). The supernatant was collected by centrifugation (16, 200 × g, 10 min), and the protein concentration was measured through a bicinchoninic acid protein assay kit (Beyotime, P0012S). An aliquot of 50 μg protein was separated via SDS-PAGE, transferred to a nitrocellulose membrane, and then blocked with 5% nonfat milk in phosphate-buffered saline (PBS, pH 7.4). The membranes were incubated with primary antibodies against ACSL4 (1:500; ABclonal, A16848), FTH1 (1:500; ABclonal, A19544), GPX4 (1:1, 000; Abcam, ab125066), COX2 (1:1,000; ABclonal, A1253), and actin (1:5, 000; ABclonal, AC026) at 4°C overnight. Blots were incubated in horseradish peroxidase-conjugated secondary antibodies against rabbit IgG (1:5, 000, CST, 7071 and 7072) for 2 h at room temperature, then subjected to chemiluminescent detection using the SuperSignal West Pico Substrate (34077, Pierce), and exposed to film. Digital images were quantified using densitometric measurements obtained using Quantity One software (Bio-Rad).