Enhanced chemiluminescence reagent

Western blotting and the analysis of the blot results was conducted as previously reported^{27 (link)}. In brief, cells were lysed and centrifuged at 12,000 g for 10 minutes at 4 °C. The supernatants were denatured at 100 °C for 5 minutes and mixed with a 1/5 volume (corresponding to the volume of the supernatants) of loading buffer (Beyotime, China). Then, 30-µg protein samples were loaded into each well in a 10% SDS-polyacrylamide gel and were subjected to electrophoresis. Once the proteins of diverse molecular weights were sufficiently separated, the proteins were transferred to PVDF membranes (Millipore, USA). Next, the PVDF membranes with the transferred proteins were blocked with 5% nonfat milk diluted in TBST for 2 hours at 25 °C and were then incubated with primary antibodies, such as SLC7A11, SLC3A2, GPX4, GSS, GCLC, ACSL4 (Cell Signaling Technology, USA) and β-actin (Beyotime, Shanghai, China), with dilutions of 1:1500, 1:2000, 1:2000, 1:2500, 1:3000 and 1:10,000, respectively, at 4 °C for 12 hours. Next, the membrane was washed again with TBST and was incubated with a 1:10,000 dilution of horseradish peroxidase (HRP)-labelled anti-rabbit IgG (Beyotime, Shanghai, China) for 1 hour at 25 °C. Finally, the respective protein bands were visualized using enhanced chemiluminescence reagents (BOSTER, Wuhan, China).

We used western blotting to detect the protein expression of target genes. Radio immunoprecipitation assay buffer mixture (Beyotime, P0013B, China) containing a protease inhibitor cocktail (Beyotime, P1006, China) was used to extract whole-cell lysates. The concentrations of these whole-cell lysates were determined using BCA protein assay kit (Beyotime, P0012, China). The whole-cell lysates from the samples were then separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis for 2 h. The proteins were transferred to 0.45-μm polyvinylidene difluoride (PVDF) membranes (Pierce Biotechnology, USA) at 400 mA for 20–40 min, wherein the timing depended on the size (kDa) of the target protein. After blocking with 5% skim milk powder in Tris-buffered saline with Tween™ (TBST) for 1 h at room temperature, the PVDF membranes were incubated with specific primary antibodies overnight at 4 °C. After washing with TBST three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (BOSTER, China) for 1 h. Finally, after treatment with enhanced chemiluminescence reagents (BOSTER, China), the images of the membranes were captured using the ChemiDoc XRS imaging system (Bio-Rad, USA).