Protease inhibitor

Cells were lysed by using RIPA buffer (Epizyme, China) containing protease inhibitor (AbMole, USA). The total proteins were separated by 10% SDS-PAGE and subsequently transferred to PVDF membranes (Millipore, USA). After blocking with 5% fat-free milk, incubate with primary antibodies against TIMP1 (A00561-1; Boster, China) and β-actin (Beyotime Biotechnology, China), respectively, using a dilution of 1 : 1000 for each antibody overnight at 4°C. After washing with TBST, secondary antibodies were introduced into the membrane for 1 h at room temperature. The interested proteins were visualized using the enhanced chemiluminescence (ECL, Epizyme), and the protein band intensity was analysed using ImageJ software.

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.