Anti slc7a11

WB was conducted according to conventional protocols. Briefly, cells treated as indicated were harvested and lysed using RIPA buffer (Beyotime, China). Proteins were extracted and transferred to nitrocellulose membranes via SDS–PAGE. Then, membranes were blocked with 5% skimmed milk for 1 h at room temperature prior to incubation with primary antibodies overnight at 4 °C. The primary antibodies used were anti-O-GlcNAc (Abcam, #ab2735), anti-OGT (Abcam, #ab184198 or #ab177941), anti-YAP (Abcam, #ab52771), anti-β-Tubulin (CST, #2128), anti-Histone-H3 (Santa Cruz, #sc-10809), anti-TFRC (Abcam, #ab84036), anti-SLC7A11 (Abcam, #175186), and anti-GAPDH (CST, #5176). For nuclear and cytosolic separation, nuclear extraction was conducted according to the manufacturer’s protocols for the Nuclear Extraction Kit (Active Motif, USA). Membranes were incubated with HRP-linked secondary antibodies [anti-rabbit (CST, #7074) or anti-mouse (CST, #7076)] for 1 h at room temperature. Signals were detected using Pierce™ ECL Western Blotting Substrate (Thermo Scientific, USA).

After sevoflurane exposure, HT22 cells were lysed in precooled RIPA lysis buffer (Cat. R0020, SolelyBio, China) with protease inhibitor (Cat. M5293, AbMole, USA). We quantified the protein using a BCA Protein Quantitation Kit (Cat. PC0020, SolelyBio, China). The equal protein in each group was separated in 10% SDS PAGE (Cat. LK303, Epizyme, China) and transferred into PVDF membrane (Cat. ISEQ00010, Millipore, USA). The primary antibodies used in this study were anti-Slc7a11 (Cat. ab175186, Abcam, dilution 1 : 1000), anti-Gpx4 (Cat. AB_2838663, Affinity, dilution 1 : 1000), and anti-GAPDH (Cat. 60004-1-Ig, Proteintech, dilution 1 : 1000). Super ECL detection reagent (Cat. SQ101, Epizyme, China) was used for visualizing the protein bands in PVDF membrane. ImageJ 1.53 software was used for quantification of the protein bands.

The smashed fresh brain tissues were selected blindly and RIPA Lysis Buffer (Beyotime, Haimen, China) and protease phosphatase inhibitors (PMSF, Beyotime) were mixed to fully ground. The protein concentration was measured using a bicinchoninic acid assay (Beyotime). The belt was transferred to the polyvinylidene fluoride membrane (Beyotime) after an electrophoresis process. Membranes were blocked with 5% skim milk blocking buffer at 37°C for 2 h and incubated with the following ferroptosis-rand BBB-related primary antibodies: anti-SLC7A11 (Abcam, Cambridge, UK; 1: 5,000), anti-GPX4 (Abcam; 1: 1,000), anti-PDGFR-β (Solarbio; 1: 1,000), anti-TFR1 (Abcam; 1: 5,000), anti-GSS (Abcam; 1: 5,000), anti-PI3K (Abcam; 1: 1,000), anti-p-PI3K (Abcam; 1: 1,000), anti-Akt (Abcam; 1: 10,000), anti-p-Akt (Abcam; 1: 1,000), and anti-β-actin (Abcam; 1: 5,000) as an internal control, at 4°C overnight in a thermostat shaker. After being washed by TBST (Tris-HCI buffer salt solution+Tween) buffer, all membranes were incubated with the second antibodies (Proteintech, Rosemont, IL, USA; 1: 5,000) at 37°C for 2 h. Immunoreactive membranes were processed with a chemiluminescence assay (Beyotime) (Wang et al., 2020 (link)). ImageJ software was used for analysis.