Tissue lysis extraction reagent

Lung tissues were homogenized with a tissue lysis/extraction reagent (Sigma-Aldrich, Carlsbad, CA, United States). The concentration of proteins in each sample was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, CA, United States). Equal amounts of cellular proteins (30 μg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated in blocking solution (5% skim milk, Millipore Co., Bedford, MA, United States), followed by overnight incubation at 4°C with the appropriate primary antibodies, as follows: anti-p65 (Cell Signaling, Denver, MA, United States), anti-p-p65 (Cell Signaling) anti-MUC5AC (Abcam), anti-AP-1 (Cell Signaling) and anti-β-actin (Cell Signaling) antibodies. The blots were washed with Tris-buffered saline containing Tween 20 (TBST), and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, United States) for 30 min at room temperature. The blots were washed again with TBST, and then, developed using an enhanced chemiluminescence kit (Thermo Scientific, San Diego, CA, United States). We evaluated the band expression values by ChemiDoc^TM (Bio-Rad, Hercules, CA, United States).

Protease and phosphatase inhibitors (Roche, Basel, Switzerland) were added to tissue lysis/extraction reagent (Sigma-Aldrich, St. Louis, MO, USA), and the lung tissue was homogenized with this solution (1/10 w/v). Protein concentrations of each sample were detected using BCA (Thermo Fisher Scientific, Waltham, MA, USA). Western blotting was performed as described previously [20 (link)], and the following antibodies were used: iNOS (1:000; Cell Signaling, Danvers, MA), COX-2 (1:000; Cell Signaling), NF-kB (1:000; 1:1000; ABCAm, Cambridge, UK), phosphor-NF-kB (1:1000; ABCAm, Cambridge, MA, USA) and MMP-9 (1:1000; ABCAm, Cambridge, MA, USA). Horse-radish peroxidase-conjugated secondary antibodies (1:10,000; Thermo Fisher Scientific, Waltham, MA, USA) were used and then detected with EZ-Western Lumi Pico (Dogenbio, Seoul, Republic of Korea). The densitometric values of each band were numerically expressed using Chemi-Doc (Bio Rad Laboratories, Hercules, CA, USA).

To identify the proteins and signaling pathways within the lung tissue, the right lung was homogenized with a tissue lysis/extraction reagent (Sigma-Aldrich). The total protein content of each sample was determined by using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). A total of 30 μg protein was electrophoresed by using 10% SDS-polyacrylamide gel and then transferred to a polyvinyl difluoride membrane. The membrane was blocked with 5% skim milk for 1 h and incubated overnight at 4 °C with the following primary antibodies: p-65 (diluted 1:1000, Abcam), phosphorylated-p65 (diluted 1:1000, Abcam), iNOS (diluted 1:1000, Abcam), HO-1 (diluted 1:1000, Cell Signaling, Beverly, MA, USA), Nrf2 (diluted 1:1000, Cell Signaling), β-actin (diluted 1:1000, Cell Signaling), lamine B1 (diluted 1:1000, Abcam). After washing three times with tris-buffered saline (TBS) that was supplemented with Tween 20, the horseradish peroxidase (HRP)-conjugated secondary antibody was diluted to 1:3000 and incubated for 1 h. Membranes were washed again, and binding was detected by using an enhanced chemiluminescence kit (Thermo-Scientific, Waltham, MA, USA). The density of each protein band was evaluated by using ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA).

Prostate tissue was homogenized with a tissue lysis/extraction reagent (Sigma‐Aldrich) and protein concentration was determined using Bradford reagent (Bio‐Rad Laboratories). Immunoblotting was performed according to methods described previously (Ko et al., 2018 (link)). Equal amounts of protein (30 μg) were heated at 100℃ for 5 min, loaded onto 10% SDS‐PAGE gels, and electrophoresed. The following primary antibodies and dilutions were used: anti‐PCNA (1:1,000 dilution, Abcam) and anti‐β‐actin (1:2000 dilution, Cell Signaling). To evaluate the proportion of protein expression, densitometric band values were determined using a Chemi‐Doc imaging system (Bio‐Rad Laboratories).