70 gam007

In order to analyse the protein levels in the liver and cultured cells, the liver tissues and cells were sonicated in ice‐cold lysis buffer and centrifuged at 14 000 g for 15 minutes. Then, the supernatant was taken, and the protein concentration was determined by the Bradford method. After the lysate was mixed with loading buffer, heated at 100°C for 10 minutes, the protein was separated on a 10% or 12% gel, and transferred to a PVDF membrane (Bio‐Rad, Hercules, CA, USA). The membranes were blocked with 5% milk in TBST (TBS containing 0.05% Tween‐20) for 1.5 hours and incubated with the following primary antibodies for overnight at 4°C: Bax (1:1000; Cell Signaling Technology, 5023S), Bcl2 (1:1000; Cell Signaling Technology, 3498S), IL‐10 (1:1000; Santa Cruz, sc‐365858), IL‐6 (1:1000; Santa Cruz, sc‐57315), NF‐κBp65(1:1000; Cell Signaling Technology, 8242S), p‐NF‐κBp65 (1:1000; Cell Signaling Technology, 3033S) and GAPDH (1:1000; Proteintech, 60004‐1‐lg). After washing 3 times with TBST, the membranes were treated with secondary antibody (1:10 000; Multi Science, 70‐GAM007 or 70‐GAR001) for 2 hours at room temperature. The signals were captured under a chemiluminescent diode X‐ray imaging system (Bio‐Rad). All experiments were repeated three times with independently prepared tissues. And the experimental results are statistically analysed.

Total proteins were extracted from the tissues or cells using RIPA buffer (89901, Thermo Scientific, USA) containing protease inhibitors (36978, Thermo Scientific, USA). The supernatant was then collected by centrifugation at 4°C for 30 min at 12,000 × g. The concentration of the protein was measured using the BCA Protein Assay Kit (P0012S, Beyotime). The proteins (50 μg per sample) were isolated on 10–12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (LC2002, Invitrogen, ThermoFisher, USA), which was blocked by 5% non-fat milk (PA201-01, BioMed, China) at room temperature for 1 h. The following primary antibodies were used for subsequent incubation: anti-PTGS2 antibody (ab15191, Abcam, UK); anti-HMGB1 antibody (ab18256, Abcam, UK); anti-GPX4 antibody (ab125066, Abcam, UK); anti-FTH1 antibody (ab183781, Abcam, UK) and anti-GAPDH antibody (ab8245, Abcam, UK). After incubation with the primary antibodies at 4°C overnight, the membrane was washed and probed by appropriate HRP-conjugated antirabbit IgG antibody (1:5,000, 7,074, Cell Signaling Technology, USA) for PTGS2, GPX4, and FTH1 or HRP-conjugated antiMouse IgG antibody (1:5,000, 70-GAM007, MultiSciences, China) at room temperature for 2 h. GAPDH served as an internal control. SignalFire™ ECL reagent (6,883, Cell Signaling Technology, USA) was used to detect the signals.