Abs118047

Total protein was extracted from BMDMs in radioimmunoprecipitation lysis buffer with a complete protease-inhibitor cocktail (Servicebio). Protein concentrations were detected using a BCA protein assay kit (Beyotime Biotechnology, Beijing, China). Protein extract was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4°C with primary antibodies against Nrf2 (AF7006; 72 kDa; 1:1000; Affinity Biologicals), TLR4 (GB11519; 95 kDa; 1:1000; Servicebio), IRF1 (abs118047; 37 kDa; 1:1000; Absin), iNOS (BA0362; 130 kDa; 1:200; Boster), arginase 1 (ARG-1; GB11285; 35-40 kDa; 1:5000; Servicebio), or GAPDH (T0004; 34 kDa; 1:5000; Affinity Biologicals). The membrane strips were then stained by incubating with an appropriate horseradish peroxidase-conjugated secondary antibody for 2 h at 25°C and visualized by enhanced chemiluminescence (Millipore, Billerica, MA, USA). ImageJ software (National Institutes of Health) was used to quantify the relative densities of proteins, which were normalized against GAPDH. All experiments were performed in triplicate.

Total and nuclear proteins were extracted from BMDMs in RIPA buffer containing protease inhibitors (Servicebio, Wuhan, China). Then protein extracts were separated by 10% SDS-PAGE and incubated overnight at 4°C with primary antibodies specific for AhR (BA2013, 96 kDa, 1:200, Boster, China), HIF-1α (PB9253, 97 kDa, 1:1,000, Boster, China), IRF1 (abs118047, 37 kDa, 1:1,000, Absin, China), NF-κB p65 (GB11142-1, 65 kDa, 1:1,000, Servicebio, China), iNOS (BA0362, 130 kDa, 1:200, Boster, China), Arg-1 (GB11285, 40 kDa, 1:5,000, Servicebio, China), H3 (GB13488, 15 kDa, 1:1,000, Servicebio, China), and β-actin (BA2305, 43 kDa, 1:1,000, Boster, China). Blottings were then probed with appropriate secondary antibodies for 2 h at 25°C and assessed via an ECL kit (Millipore, MA, USA). Triplicate analyses of all samples were conducted.