Anti β actin antibody

Cells were lysed with 100 µl of lysis buffer containing protease inhibitors (Beyotime, Shanghai, China). The lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, and then incubated with an anti-DDX43 primary antibody (H00055510-M07, Abnova) overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated goat-anti-mouse secondary antibody (sc-2005, Santa Cruz Biotechnology) was used. The protein bands were detected by enhanced chemiluminescence detection. Anti-β-actin antibody (AA128, Beyotime, Shanghai, China) was used as a loading control.

Mice macrophages were prepared in 12-well plates as described above and incubated with and without peptides for 2 h at 37 °C. After 2 h incubation, cells were washed with Opti-MEM twice and infected with SC19 at a MOI of 5 for 0, 15, 30, 60, and 120 min to determine different protein expression. Then, the macrophages were lysed by the RIPA buffer with PMSF (Beyotime, Beijing, China) and concentrations were determined using a BCA protein detection kit (Beyotime). The cell lysates were separated by a 10–15% SDS-PAGE gel and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were immunoblotted with anti-NF-κB p65 Ab (Bioss, Beijing, China), anti-phospho-NF-κB p65 Ab (Beyotime), anti-ERK1/2 Ab (Bioss), anti-phospho-ERK1/2 Ab (Cell signaling technology, Danvers, MA, USA), anti-TLR2 Ab (Wanlei Life Sciences, Shenyang, China), anti-TLR4 Ab (Santa Cruz Biotechnology, Inc.), and anti-β-actin antibody (Beyotime).

SAA was provided by Shandong Target Drug Research Co. Ltd (Shandong, China). Prednisone acetate was the product of Zhejiang Xianju Pharmaceutical Co. Ltd. (Zhejiang, China). Doxorubicin hydrochloride for injection was produced by Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China). The antibodies used in this study were anti-NF-κB p65 (ab16502, Abcam), anti-inhibitor of NF-κB (IκB) α (ab32135, Abcam), anti-phosphorylation-IκBα (p-IκBα, Ser-36, ab133462, Abcam) and anti-podocin (sc-21009, Santa Cruz Biotechnology, Inc.). Goat anti-rabbit IgG was also the product of Abcam. BCA protein assay kit, anti-β-actin antibody, HRP-labeled goat anti-mouse IgG (H+L) and lipid peroxidation malonaldehyde (MDA) assay kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Superoxide dismutase (SOD) detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Cells were collected and lysed in 1х SDS loading buffer containing protease inhibitors. Protein lysates were separated using 12% SDS-PAGE and subsequently transferred to polyvinylidene fluoride membrane (Millipore, USA) following by blocking with QuickBlock™ blocking buffer (Beyotime, China) and incubation with antibodies directed against GPX4 (1:1000, 52,455, CST, MA, USA) or NRF2 (1:800, sc-365,949, Santa cruz) overnight at 4℃. Anti-β-actin antibody (1:5,000, Beyotime, China) served as control. After incubation with a secondary antibody, ECL (Beyotime, China) was used to amplify the binding antibody signal, and images were obtained with a UVITEC photo documenter. The full gel images in the paper figures are displayed in Figure S1.

For immunoprecipitation (IP), cells were suspended in lysis buffer (50 mmol/L Tris-Cl (pH 7.4), 150 mmol/L NaCl, 5% glycerol, 1% NP-40, 1 mmol/L EDTA), supplemented with 1 mM PMSF and 4 μg/mL protease inhibitor cocktail (Sigma). Lysates were centrifuged at 13,000 g for 10 min, and the supernatant was added to 2 μL indicated antibodies and 100 μL protein A agarose beads (Invitrogen) to incubate for 4 h at 4 °C. Afterward, protein A beads were washed with 250 mmol NaCl four times. For western blot, total proteins of liver tissue and LO2 cells were extracted with extraction buffer (RIPA). Nuclear proteins were extracted with the Nucleoprotein Extraction Kit protocol (Shanghai Sangon Biotech, China). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into a PVDF membrane (Millipore). The membranes were incubated with 5% BSA to block other contaminants, and then with primary antibodies. Immunoblotting was performed using anti-Bdh1 antibody (Abcam, UK), anti-Nrf2 antibody and anti-Histone H3 antibody (CST, USA), anti-cleaved caspase 3 antibody, anti-IL-18 antibody, anti-IL-1β antibody, anti-GAPDH antibody, and anti-β actin antibody (Beyotime, China).