T57046

The total proteins were lysed using a lysate buffer (Beyotime, Shanghai, China) for 30 minutes and washed with PBS. Polyacrylamide gel electrophoresis was employed to separate the protein, which was then loaded onto a polyvinylidene fluoride membrane (Millipore). Non-specific antigens were blocked with 5% skim milk for 2 h, followed by immunoblotting with different antibodies, including GSDME (1:1000, ab215191, Abcam), cleaved Caspase-3 (1:1000, #9664, CST), Glutathione Peroxidase 4 (1:1000, ab125066, Abcam), Transferrin Receptor (1:1000, ab214039, Abcam), xCT (1:1000, T57046, Abmart), GAPDH (1:5000, 60004-1-Ig, Proteintech).

All antibodies used in this study were listed below: Anti-CYLD (ab137524, abcam), Anti-ACSL4 (ab205199, abcam), Anti-TFRC (ab214039, abcam), Rabbit monoclonal Anti-YAP (ab205270, abcam), Mouse monoclonal Anti-YAP (66900-1-Ig, Proteintech), Anti-GAPDH (92310SF, CST), Anti-ki67 (28074-1-AP, Proteintech), anti-cyclin D1 (WL01435a), anti-SLC7A11 (T57046, abmart), anti-FSP1 (T55799, abmart), anti-GCH1 (MG880265, abmart) and anti-GPX4 (ab262509, abcam).

Cells were collected and used to extract the total protein with the mixture of the radioimmunoprecipitation (RIPA) lysis buffer, the protease inhibitor cocktail (1:50) and phenylmethanesulfonyl fluoride (PMSF) (1:100). The protein concentrations were quantified using a Bradford Protein Assay Kit (Proandy, China). The same amount of protein (30 µg) was separated by 10–12% SDS-PAGE electrophoresis followed by transferring onto the polyvinylidene difluoride (PVDF) membranes. Then the membranes were blocked in 5% non-fat skim milk at room temperature for 1 h. After three times washes with Tris-HCl buffered saline with Tween-20 (TBST), these membranes were put into the corresponding primary antibodies and incubated overnight at 4 ℃. The primary antibodies included GPX4 (1:1000, T56959, Abmart, China), Nrf2 (1:3000, 16396-1-AP, Proteintech, China), FTH1 (1:1000, PTM-6761, PTMBio, China), SLC7A11 (1:1000, T57046, Abmart, China), GAPDH (1:5000,10494-1-AP, Proteintech, China). The next day, the membranes were washed with TBST and put into the HRP-conjugated secondary antibody (1:5000, SA00001-2, Proteintech, China) at room temperature for another 1 h. Finally, the images could be captured by enhanced chemiluminescence reagent (ECL, Beyotime Biotechnology, China) and chemiluminescent imager (Tanon-5200, China).