A2413

Proteins were extracted and analyzed by western blotting according to standard protocols using primary antibodies specific for p53 (#A18803, ABclonal), SLC7A11/xCT (#A2413, ABclonal), and GAPDH (#AC033, ABclonal).

The cells were cleaned with 1 × PBS before being lysed with M-PER mammalian protein extraction reagent (78501, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with a Cocktail (11873580001, Roche, Basle, Switzerland). Protein concentrations were measured as indicated by the manufacturer using the BCA protein assay kit (23250, Thermo Fisher Scientific, Waltham, MA, USA).
Protein samples (30 μg) were separated in SDS-PAGE gels ranging from 4% to 15% and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, San Francisco, CA, USA). After blocking in 5% skim milk for 60 min, the membranes were treated with the appropriate primary antibodies and fluorescent-conjugated secondary antibodies. Primary antibodies against SRSF2 (ab204916, 1:1000, Abcam, Cambridge, UK), SLC7A11 (A2413, 1:1000, ABclonal, Wuhan, China), ACSL4 (A6826, 1:1000, ABclonal, Wuhan, China), and GPX4 (A1933, 1:1000, ABclonal, Wuhan, China) were used. The internal standard was probed with an anti-β-actin-peroxidase monoclonal antibody (A3854, Sigma-Aldrich, Bedford, MA, USA). In a Tanon 5200 MultiImage System (Tanon Science & Technology, Shanghai, China), ECL reagents (34080, Millipore, Burlington, MA, USA) were used to observe the bands. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to calculate band intensities.

The samples were placed in the tissue fixative (Meilunbio, China) for more than 24 hours. The pathological tissue samples were dehydrated and transparent, dipped in wax, and embedded in sequence. Paraffin sections of 3 mm samples were prepared on a paraffin microtome. The paraffin sections were sequentially subjected to dewaxing and hydration, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB color development, hematoxylin staining, hematoxylin reverse blue, and dehydration operations. Added 5-10 µL of neutral resin dropwise (Sinopharm Chemical Reagent, China) to the stained area and mounted the sections. The stained sections of pathological tissue samples were placed under a slide scanner for scanning, and quantitative statistical analysis was performed. The IHC score was calculated by combining the ratio score (percentage of positively stained cells) with the staining intensity score. The scale and staining intensity scores were then multiplied to generate a score for each case. The antibody information used in IHC assay, Anti-MKL-1 (77098, CST, USA) Anti-SLC3A2 (A3658, ABclonal, China), Anti-SLC7A11 (A2413, ABclonal, China).