Anti xct

Brain tissues and isolated primary microglia were lysed in SDS sample buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 2 mM ethylenediaminetetraacetic acid). Lysates were subjected to SDS-PAGE and then transferred onto a PVDF membranes (Hybond-P; GE Healthcare). The membrane was blocked with a 3% skim milk/PBS-Tween 0.5% (PBST) solution. The blot was then placed in the PBST solution containing anti-xCT (Abcam, Cambridge, UK), anti-Iba-1 (Abcam), anti-connexin 43 (BD Transduction Laboratories, Lexington, UK) or anti-β-actin (Cell Signaling Technology, Danvers, MA). Immunoreactive signal was detected using HRP-linked anti-rabbit or mouse IgG and ECL detection reagents (GE Healthcare) following the manufacturer’s specifications. The protein content of each sample was measured using the bicinchoninic acid (BCA) protein assay system (Thermo Fisher Scientific). The density of the immunoblot signal was semi-quantified using Image J software (National Institutes of Health, Bethesda, MD).

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).