Horseradish peroxidase hrp conjugated anti rabbit or anti mouse igg

Diosmetin, purity >98%, was purchased from Chengdu Pufei De Biotech Co., Ltd. Dexamethasone was purchased from TianJin KingYork Group HuBei TianYao Pharmaceutical Co., Ltd. LPS (Escherichia coli 055:B5) and Dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co (St. Louis, MO, USA). Penicillin and streptomycin, foetal bovineserum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) for cell culture use were purchased from Invitrogen-Gibco (GrandIsland, NY, USA). MPO and MDA determination kit were provided by the Jiancheng Bioengineering Institute of Nanjing (Jiangsu, China). Mouse TNF-α, IL-1β and IL-6 ELISA kits were provided by Biolegend (San Diego, CA, USA). Antibodies against NLRP3, ASC, caspase-1, IL-1β, Nrf2, HO-1 and β-actin were purchased from Cell Signaling (Boston, MA, USA). The horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG were obtained from protein-tech (Boston, MA, USA). All other chemicals, unless specifically stated elsewhere, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Snapped frozen liver tissues were treated with RIPA lysis buffer for protein extraction (Beyotime, Nanjing, China). Protein concentrations were determined using the BCA assay (Beyotime, Nanjing, China). Proteins (60 µg) were loaded into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk at room temperature and incubated overnight at 4 °C with primary antibodies against NLRP3 (1:1,000; Cell Signaling Technology, USA), ASC (1:1,000; Santa Cruz, USA), Cleaved caspase1 (1:1,000; Santa Cruz, USA), Bcl-xl (1:1,000; Cell Signaling Technology, USA), Bax (1:1,000; Cell Signaling Technology, USA), Cleaved-caspase3 (1:1,000; Cell Signaling Technology, USA) and GAPDH (1:2,000; Proteintech, China). After 3 washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (1:10,000; Proteintech, China) at room temperature for 1 h. The proteins bands were developed using Electrochemiluminescence (Biorad, USA) reagents and imaged using the Imaging System (Peiqing, Shanghai, China). The gray value was quantified using Image J software.