Ecl western blotting detection

Lung fragments (0.05 g) were homogenized in ice-cold RIPA lysis buffer (Beyotime, China), centrifuged at 14,000 rpm for 15 min, and the supernatant was collected. Protein concentrations were determined by the BCA protein assay kit (Beyotime, China). Polyacrylamide gel (10%) electrophoresis was used to isolate the proteins. The proteins were then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The nitrocellulose membrane was probed with anti-Toll-2, anti-ERK, anti-Toll-9, or anti-IDO monoclonal antibodies at 4°C overnight. The next day, the membrane was incubated with horseradish peroxidase-labelled goat anti-rabbit IgG antibody (1 : 1000) at room temperature, followed by alkaline phosphatase-conjugated secondary antibodies (BD, USA) for 2 h at room temperature on a shaker. The protein bands were displayed by ECL™ western blotting detection (GE healthcare, No. RPN2106) and quantified by Gel-Pro imaging software.

Cells were washed 2 times with ice-cold PBS and lysed in RIPA buffer containing 10 mg/ml of aprotinin, PMSF, and leupeptin (Sigma-Aldrich), 5 mM β-glycerophosphate, 50 mM NaF, and 0.2 μM sodium orthovanadate for 30 minutes at 4 °C, followed by centrifugation at 13.000 g for 15 minutes; supernatants were then collected and kept frozen at −20 °C. Total protein concentrations were determined by Bradford protein assay using a Bio-Rad protein assay kit. Standardized amounts of protein samples (25 μg to 50 μg) were separated by 10% SDS-PAGE. Proteins were subsequently transferred onto nitrocellulose membrane and incubated in blocking solution (PBS, 5% nonfat milk, 0.2% Tween-20) for 1 hour at room temperature followed by overnight incubation with the NAIP-J236 (link) rabbit polyclonal antibody at 4 °C diluted at 1:1500. Membranes were washed with PBS-T (PBS, and 0.2% Tween-20) 3 times followed by incubation with secondary antibody (anti-rabbit or mouse; Cell Signaling) for 1 hour at room temperature at the dilution suggested by the manufacturer. Loading control antibody complexes were visualized by autoradiography using the ECL Plus and ECL Western Blotting detection systems (GE Healthcare). Quantification was performed by scanning the autoradiographs, and signal intensities were determined by densitometry analysis using the ImageJ program (National Institutes of Health, USA).

Protein was extracted by using RIPA Buffer according to the manufacturer’s standard protocol (Cell Signaling Technology). Determination of the concentration of proteins was performed by using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) following its protocol. After gel electrophoresis and blotting on a Whatmann Protran BA85 membrane (GE Healthcare), membranes were incubated with primary antibody rabbit anti-BRG1 (1:2500, Santa Cruz) overnight. Incubation with a secondary antibody anti-Rabbit (1:2000, Promega) for 1h was followed by developing the membranes using ECL™ Western Blotting Detection (GE Healthcare) as detection solution.