Bicinchoninic acid assay kit

Protein levels were assessed by Western blotting. Antibodies used to detect the following proteins are shown as catalogue numbers in parentheses: Bax (33-6400, Thermo Fisher Scientific Ltd., Waltham, MA); Bcl-2 (MA5-11757, Thermo Fisher Scientific Ltd., Waltham, MA); caspase-3 (MA1-91637, Thermo Fisher Scientific Ltd., Waltham, MA); p-Akt (9271, Cell Signaling Technology, Shanghai, China); Akt (9272, Cell Signaling Technology, Shanghai, China); and PI3K (4292, Cell Signaling Technology, Shanghai, China). A bicinchoninic acid assay kit (7780, Cell Signaling Technology, Shanghai, China) was used to measure protein concentrations prior to gel electrophoresis. Proteins were separated via 10% (w/v) sodium dodecyl sulphate–polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes; the membranes were blocked with 5% (w/v) blocking solution (non-fat milk) and incubated in blocking buffer with the primary antibodies overnight at 4 °C. Goat secondary antibodies conjugated with horseradish peroxidase were added, and a chemiluminescence kit was used to detect proteins.

Total proteins in the lungs were extracted using protein lysis buffer (20 mM Tris-HCL, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM Sodium pyrophosphate, 1.22 mM MgSO₄, 20 mM Tris-HCL, and 150 mM NaCl) containing protease inhibitor cocktail (MilliporeSigma, Burlington, MA, USA). Protein concentration was determined using a bicinchoninic acid assay kit (Cell Signaling Technology, Danvers, MA, USA). Aliquots of protein extract (40 μg per lane) were separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Burlington, MA, USA), and then blocked with 5% bovine serum albumin in TBST (mixture of Tris-buffered saline and 0.1% Tween 20) for 1 h. Then, the membranes were incubated overnight at 4 °C with primary antibodies against p65, p-p65, p-IKKα/β, IκBα or β-actin (all at 1:1000, Cell Signaling Technology, Danvers, MA, USA), COX-2 (1:200, Cayman Chemical, Ann Arbor, MI, USA), NOX1 or NOX4 (both at 1:500, Novus Biologicals, Littleton, CO, USA), or NOX2 (1:100, Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation with anti-mouse or anti-rabbit secondary antibody for 1 h at room temperature. The bands were visualized with the enhanced chemiluminescence Plus detection reagent using the ChemiDoc XRS+ system (Bio-Rad), and quantified using the NIH Image J software.