Anti gpx4

tert-Butyl hydroperoxide, cumene hydroperoxide, hydrogen peroxide, ferrostatin-1, cyclosporine A, GSH, GSSG, and MG132 were from Sigma. Doxorubicin hydrochloride, liproxstatin-1, deferoxamine, mitoQ, and SKQ1 were from Cayman Chemical. MitoPeDPP and mito-FerroGreen were from Dojindo. Necrostatin-1s was from Cell Signaling Biotechnology. MitoSOX, propidium iodide, and Hoechst 33,342 were from Invitrogen. The following antibodies were used: anti-HMGB1 (3935), anti-HO-1 (82,206), anti-VDAC (4661), anti-catalase (14,097), and anti-GAPDH (2118) from Cell Signaling Biotechnology; anti-Bach1 (sc-271211) from Santa Cruz Biotechnology; Anti-FTMT (PAD251Mu01) from Cloud-Clone Corp.; anti-GPX4 from R&D Systems.

Total protein was isolated with RIPA buffer from cultured cells and exosomes, heated at 95 °C for 10 min and quantified using a NanoDrop2000 spectrophotometer. Each sample was separated on 10% SDS–PAGE gels and transferred onto PVDF membranes (Roche, Basel, Switzerland). Then, membranes were blocked for 1 h and incubated with primary antibodies, including anti‐MTP (1:500; Santa Cruz, sc‐135994), anti‐ACSL4 (1:500; Santa Cruz, sc‐365230), anti‐PRAP1 (1:1000; Proteintech,11932‐1‐AP), anti‐xCT (1:1000; Abcam, ab175186), anti‐GPX4 (1:1000; R&D,#565320), anti‐ZEB1 (1:1000; CST; #70512), anti‐CD9 (1:1000; CST; #13403), anti‐Alix (1:1000; CST; #92880), anti‐CD69 (1:1000; CST; #28633), anti‐TSG101 (1:500; Santa Cruz, sc‐7964) and anti‐GAPDH (1:3000, Santa Cruz, sc‐32233), for at least 16 h at 4 °C. Then, the membranes were incubated with the corresponding secondary antibodies at a 1:5000 dilution for 1 h at room temperature. All samples were normalized to GAPDH.