Slc7a11

Protein extraction of neuronal cells in the control, M0-Exosome, and M1-Exosome groups was carried out using RIPA (Beyotime, Shanghai, China). Protein concentration was determined using a BCA assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. For each group, protein (20 μg/lane) was loaded and separated by 12% SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA). The separated proteins were then transferred to PVDF membranes with a current of 300 mA for 80 min, blocked using 3% bovine serum albumin (Beyotime, Shanghai, China) for 2 h, and incubated overnight with primary antibodies against GPX4 (1:1000; Abcam), SLC7A11 (1:1000; ABclonal), FTH1 (1:1000; ABclonal), and β-actin (1:5000; ABclonal). Subsequently, the membranes were washed three times in TBST and incubated in HRP-conjugated secondary antibody (1:4000; goat anti-rabbit IgG, ABclonal) for 1 h. The membranes were once again washed in TBST. The protein bands were then exposed to the ECL reagent (Beyotime, Shanghai, China) and visualized using a chemiluminescent detector (Tanon Science and Technology Co., Ltd.). The relative protein expression was quantified using ImageJ software (National Institutes of Health) with β-actin as the control.

The liver tissue or cells were prepared to homogenate with cooled PIRA lysate. After centrifugation, the supernatant was collected and the total protein concentration was measured. The 30 μg proteins were separated by SDS-PAGE electrophoresis, and transferred to polyvinylidene fluoride (PVDF) membranes. The blots were incubated with 5% skimmed milk. After washed with TBST, the blots were incubated with primary antibody of Collagen I (Servicebio, GB112543), Collagen III (Servicebio, GB11023), NADPH Oxidase 4 (NOX4, Boster, A00403), 4-Hydroxynonenal (4-HNE, Arigo, ARG23717), Transferrin Receptor 1 (TfR1, Invitrogen, 13–6800), Divalent Metal Transporter 1 (DMT1, Absin, abs112967), Ferroportin 1 (FPN1, Alpha Diagnostic International, MTP11-A), Glutathione Peroxidase 4 (GPX4, Huabio, ET1706-45), the glutamate/cystine antiporter solute carrier family 7 member 11 (SLC7A11, Abclonal, A2413), Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2, Servicebio, GB11962), Heme Oxygenase-1 (HO-1, Servicebio, GB12104), GAPDH (Servicebio, GB15002), α-Tubulin (GeneTex, GTX628802) overnight at 4°C. The second day, blots were incubated with HRP-conjugated secondary antibodies at room temperature. Finally, enhanced chemiluminescent (ECL) method and Image J software were respectively used to detect the immunoreactive protein and analyze the mean gray value.

Western blot analysis was conducted according to a previous study [41 (link)]. The weighed liver samples were added to an equal proportion of RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and the supernatant was obtained after ultrasonic crushing and centrifugation. A BCA kit (Beyotime Biotechnology, Shanghai, China) was used for protein concentration measurement. Then, protein samples were separated by SDS-PAGE, transferred to a membrane, and sealed. The membrane was incubated with a specific primary antibody of PTGS2, ACSL4, or SLC7A11 (Abclonal, Wuhan, China) or GAPDH (Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. Fluorescent signals were detected with an imager (Chemidoc-MP imaging system, BioRad, Hercules, CA, USA), and the protein quantification results were generated by Image J 1.53a software (National Institutes of Health, Bethesda, MD, USA).