Pro il 1β

Fibroblast levels of relevant proteins were assessed by Western blot as previously described (39 (link)). Briefly, fibroblasts were lysed and homogenized with lysis buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM NaF, 1 mM Na₃VO₄, 10 mg/mL leupeptin, 10 mg/mL aprotinin, and 20 mM PMSF]. Proteins from each sample were separated on 10–15% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 3% bovine serum albumin (Wisent Inc, St-Bruno, QC, Canada), the membranes were probed with primary antibodies against mouse NLRP3, caspase 1 p20 (Adipogen, San Diego, CA, USA) at 1:500 dilution; pro-IL-1β (R and D Systems, Minneapolis, MN, USA) at 1:500 dilution; nitrotyrosine (Santa Cruz, Dallas, TX, USA) at 1:1,000 dilution; and β-actin (Santa Cruz, Dallas, TX, USA) at 1:2,000 dilution. After treatment with appropriate secondary antibodies, the specific bands were detected with an enhanced chemiluminescence (ECL) system and quantified with an imaging densitometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Lung tissues were homogenized in RIPA in the presence of a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and Phosphatase Inhibitor Cocktail (Roche). The protein concentrations were determined by a BCA kit (Pierce, Rockford, IL, USA). Equal concentrations of protein (25 μg) were separated by SDS-PAGE on 12% acrylamide gels. The primary antibodies used were as follow: NLRP3 (Adipogen, San Diego, CA, USA), caspase 1 (p20) (Adipogen), IL-1β, pro-IL-1β (R&D Systems, Minneapolis, MN, USA), TLR2 (Millipore, Billerica, MA, USA), AANAT (Sigma), ASMT (Abcam, Boston, MA, USA) and GAPDH (KANGCHEN Biotech, Shanghai, China) for 1 h at 37°C, followed by at 4°C overnight. Blots were washed thrice with TBST and probed with appropriate secondary antibodies for 1 h at room temperature. After washing three times, the immune-reaction was analyzed using the ECL detection system (Pierce). The band density was determined by ImageJ software (NIH, Bethesda, MD, USA).