Micro bicinchoninic acid protein assay kit

Following treatment or transfection, MSC were washed with PBS and lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 120 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.1% Triton) in the presence of phosphatase and protease inhibitors on ice for 30 minutes. Cell debris was pelleted by centrifugation for 10 min at high speed. Protein concentration was quantified using a micro bicinchoninic acid protein assay kit (Thermo Fisher Scientific Inc). Proteins from control and treated cells were separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were electrotransferred to polyvinylidene difluoride membranes, which were then blocked overnight at 4°C with 5% non-fat dry milk in Tris-buffered saline (150 mM NaCl, 20 mM Tris–HCl, pH 7.5) containing 0.3% Tween-20 (TBST). Membranes were further washed in TBST and incubated with primary antibodies directed against COX-2 (1/10,000), IκB, phosphorylated IκB (1/1,000), or GAPDH (1/1,500). Washing was then performed in TBST, followed by a 1 hour incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1/10,000) or anti-mouse IgG (1/5,000) in TBST containing 5% non-fat dry milk. Immunoreactive material was visualized by Western Lightning Enhanced Chemiluminescence Pro reagents (Perkin Elmer).

Bacterial cells were grown as described above. The cells from a 1.5-liter culture were harvested by centrifugation at 6,000 × g for 10 min at 4°C and washed, and E. coli and P. aeruginosa cells were suspended in 50 mM phosphate buffer (pH 7.0). For H. influenzae, S. aureus, and S. pneumoniae, KPN buffer (20 mM potassium phosphate, 140 mM NaCl [pH 7.5]) was used. Cells of S. aureus were first treated with lysostaphin (400 μg/ml) for 1 h at 37°C before addition of 3 μg/ml of a protease inhibitor cocktail (Sigma-Aldrich), DNase (6 μg/ml), and RNase (6 μg/ml). For S. pneumoniae and H. influenzae, lysozyme (400 μg/ml) was also added. After 30 min of treatment, cells were disrupted by a French press instrument, and the bacterial lysates were centrifuged at 6,000 × g for 30 min at 4°C to remove unbroken cells. The supernatant was then centrifuged at 150,000 × g for 40 min at 4°C using a fixed-angle rotor to collect the membranes. The membranes were suspended in a minimal volume of buffer (typically 500 μl) and stored at −80°C. The protein concentrations in the membrane preparations were estimated by using the Micro bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL) with bovine serum albumin as a standard.

Lungs were washed three times with PBS to obtain BAL, and the cell-free supernatants were analyzed for IL-2, IL-4, IL-5, IL-9, IL-10, IL-12, IL-13, IL-17, amphiregulin, TNF-α, GM-CSF, and IFN-γ by enzyme linked immunosorbent assay using methods recommended by the manufacturers (BD Biosciences, eBioscience, and R&D Systems). Levels of albumin in BALF were determined with the bacillus Calmette–Guérin albumin assay kit (Sigma-Aldrich). Total protein concentrations in BALF were analyzed using a Micro bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Nitrite levels were quantified using a colorimetric Griess reagent kit (Life Technologies). The critical commercial kits used here are mentioned in SI Appendix, Table S5.

Following treatments or transfection, HT1080 cells were washed with phosphate-buffered saline and lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 120 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.1% Triton) in the presence of phosphatase and protease inhibitors on ice for 30 minutes. Cell debris was pelleted by centrifugation for 10 minutes at high speed. Protein concentration was quantified using a micro bicinchoninic acid protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Proteins from control and treated cells were separated by SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were electrotransferred to polyvinylidene difluoride membranes, which were then blocked overnight at 4°C with 5% nonfat dry milk in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl, pH 7.5) containing 0.3% Tween-20 (TBST). Membranes were further washed in TBST and incubated with primary antibodies directed against MT1-MMP (1/10 000), p105, phosphorylated p105, Erk, phosphorylated Erk, IκB, phosphorylated IκB (1/1000), or GAPDH (1/1500). Washing was then performed in TBST, followed by a 1-hour incubation with horseradish peroxidase–conjugated anti-rabbit IgG (1/10 000) or anti-mouse IgG (1/5000) in TBST containing 5% nonfat dry milk. Immunoreactive material was visualized by Western Lightning Enhanced Chemiluminescence Pro (Perkin Elmer, Waltham, MA, USA).