Anti gpx4

Recombinant purified TNFα, the annexin V-FITC antibody and the 7-AAD antibody were purchased from BioLegend (London, United Kingdom). Necrostatin-1_s (Nec-1_s) was obtained from BioVision, Inc. (Milpitas, CA, USA). The zVAD-fmk (herein referred to as zVAD) was purchased from Bachem (Weil, Germany). Erastin (era), GSK’872, Necrosulfonamide (NSA) and the anti-MLKL antibody (clone 3H1) were obtained from Calbiochem (Merck Millipore, Darmstadt, Germany). Synthesis of the ferrostatin derivative 16-86 and the ferroptosis inducer RSL3 were described previously [12 (link), 14 (link)]. Ferrostatin-1 (Fer-1) was purchased from Xcess Biosciences Inc. (San Diego, CA, USA). Dabrafenib was purchased from Selleckchem (Absource Diagnostics, Munich, Germany). GW806742X was purchased from Biomol (Hamburg, Germany). The monoclonal anti-ACSL4, anti-GPX4, the anti-human and anti-mouse monoclonal phospho-MLKL antibodies were all obtained from Abcam (Cambridge, United Kingdom). The β-actin antibody was purchased from Cell Signaling (Frankfurt, Germany).

T24 and MB49 cells (10⁶ per dish) subjected to the indicated treatments were harvested and lysed in lysis buffer (M-PERTM mammalian protein extraction reagent, Thermo Fisher Scientific Inc.) for 30 min on ice and then centrifuged at 14,000 rpm for 20 min at 4 ℃ to remove precipitates. The obtained proteins were adjusted to equal loads (20 µg per well). They were separated through electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide gel and subsequently transferred to a 0.45 μm polyvinylidene difluoride (PVDF) membrane, blocked using 5% skim milk, and immunoblotted using anti-GPX4 (cat no. #ab125066, 1:3000 dilutions, Abcam plc.) and GAPDH (cat no. #2118, 1:5000 dilutions, Cell Signaling Technology Inc.) monoclonal antibodies. The membranes were then washed using Tris-buffered saline supplemented with 0.1% Tween-20 and incubated again with horseradish peroxidase (HRP)-conjugated secondary antibody (cat no. #7074, 1:5000 dilutions, Cell Signaling Technology Inc.). The corresponding bands were detected using HRP substrate (Merck Millipore) and captured using an imaging system (UVP Bio-Spectrum; Analytik Jena US LLC, Upland, CA). The images were analyzed by ImageJ software (NIH, Bethesda, Maryland) for protein expression normalization and quantification.

Cell extracts and tissue homogenates were lysed on ice with RIPA buffer (Sigma Aldrich, St. Louis, Missouri, USA), including DTT and protease inhibitors (Protease and Phosphatase Inhibitor Mini Tablets, Thermo Scientific, Waltham, MA, USA). Proteins, in the amount of 30–50 μg, were subjected to SDS PAGE on 4%–12% denaturing gel and probed with the following antibodies: anti‐ASIC1a (1:1000 Abcam, Cambridge, UK), anti‐GPx4 (1:1000, Abcam, Cambridge, UK), and anti‐GAPDH (1:1000, Sigma Aldrich) as a loading control. Horseradish peroxidase‐conjugated goat anti‐mouse or goat anti‐rabbit IgG (1:10,000, Applygen) was used as the secondary antibody, and the signal was visualized using Super ECL Plus substrate (P1050, Applygen). The indicated proteins were quantified with Image J software.
All of the uncropped blots of these western blot experiments were presented in the Data S1.

Cells were lysed using lysis buffer (Promega Corp.) supplemented with a complete protease inhibitor cocktail (Roche Applied Science) and phenylmethylsulfonyl fluoride (Sigma-Aldrich Corp.). Tissues were further homogenized using 0.5 mm glass beads in a Bullet Blender high-throughput homogenizer (Next Advance, Inc.). After pelleting cellular debris by spinning at 16 000 rpm at 4°C for 15 min, protein was quantified by Bradford assay (66 ). For western blotting, 40–50 µg protein was added per lane of 4–12% Bis-Tris gels (Invitrogen Corp.). Primary antibodies were diluted in Odyssey blocking buffer (LI-COR Biosciences). Antibodies used included: anti-FXN (provided by Franco Taroni M.D., Istituto Besta), anti-HO1 (# sc-10789, Santa Cruz Biotechnology, Inc.), anti-NQO1 (#3187, Cell Signaling Technologies), anti-Gpx4 (#ab125066, AbCam), anti-nrf2 (sc-25820, Santa Cruz) and anti-actin (#A2668, Sigma). Direct conjugated secondary antibodies (anti-rabbit IRdye800Cw and anti-mouse IRdye680 from LI-COR) were used to detect and quantify the signal of primary antibodies and imaged using a LI-COR Odyssey.

DL-homocysteine (Hcy, H4628) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Rituximab (RTX, 10 mg/ml), a monoclonal anti-human CD20 antibody, was purchased from Roche (Basel, Switzerland). Ferrostatin-1 (Fer-1, S7243) and liproxstatin-1 (lip-1, S7699) were purchased from Selleckchem (Houston, TX, USA). The following antibodies were used in this work. Anti-β₂GPI (bs-1570R) was purchased from Bioss Inc. (Beijing, China). Anti-CBS (cystathionine β-synthase, 14787-1-AP) and anti-CSE (cystathionine γ-lyase, 12217-1-AP) were purchased from Proteintech (Rosemont, IL, USA). Anti-CD31 (sc-18916), anti-nephrin (sc-377246) and anti-PDGFRβ (platelet-derived growth factor receptor beta, sc-374573) were purchased from Santa Cruz Biotech (CA, USA). Anti-4-HNE (4-hydroxynonenal, MAB3249) was purchased from R&D Systems (MN, USA). Anti-xCT (catalytic subunit of cystine/glutamate antiporter System Xc-, also called SLC7A11, ab37185), anti-LOX15 (Lipoxygenase 15, ab23691), anti-GPX4 (glutathione peroxidase 4, ab125066), and anti-TFR (transferrin receptor, ab214039) were purchased from Abcam (Cambridge, MA, USA). Anti-TF (transferrin, A1448), anti-SLC40A1 (A14884), anti-β-actin (AC038), HRP-conjugated goat anti-rabbit (AS014), and HRP-conjugated goat anti-mouse (AS003) were purchased from ABclonal (Wuhan, China).

Anti-Gpx4 (Abcam, ab41787, 1:2000), Anti-xCT (Abcam, ab37185, 1:2000), Anti-Cas9 (Cell signaling, 14697, 1:1000,), Anti-ß-Actin (Sigma, A1978, 1:10,000), Anti-GCLC (Santa Cruz, sc-166345, 1:1000), Anti-Ascl1 (BD Pharmingen, 556604, 1:1000), Anti-Txnrd1 (Cell signaling, 15140S, 1:1000), Anti-Txnrd2 (Cell signaling, 12029, 1:1000), Anti-Acsl4 (Santa Cruz Biotechnology, sc-271800, 1:2000), Anti-Txn1 (Cell signaling, 2429S, 1:1000), Anti-GAPDH (Cell signaling, 97166S, 1:2000), Anti-FSP1 (previously described⁵⁵ , kindly provided by M. Conrad, undiluted hybridoma supernatant), Anti-REST1 (ThermoFisher, BS-2590R, 1:1000), Anti-REST1 (Abcam, ab21635, 1:1000), Anti-TXNIP (Cell signaling, 14715S, 1:1000), Anti-CD71 (Santa Cruz, sc-65882, 1:2000), Anti-cMyc (Abcam, ab32072, 1:2000), Anti- NCAM (Invitrogen, PA5-79717, 1:1000), Anti-Vimentin (Abcam, ab137321, 1:1000), Anti-YAP1 (Cell signaling, #4912, 1:1000), Anti-Synatophysin (Invitrogen, MA5-14532, 1:1000), Anti-AGPAT2 (Thermo Fisher, PA5-76010, 1:2000), Anti-AGPAT3 (Thermo Fisher, PA5-101343, 1:2000). HRP-conjugated secondary antibodies: goat-anti-mouse-HRP (Linaris GmBH, 20400-1 mg, 1:10,000), goat-anti-rabbit-HRP (Linaris GmBH, 20402-1 mg, 1:10,000), goat-anti-rat-HRP (Sigma, A9037-1 ml, 1:10,000).