Irdye conjugated goat anti mouse or goat anti rabbit antibodies

Cells were washed twice with cold PBS (pH 7.4) and RIPA lysis buffer mixed with phosphatase inhibitor, and phenylmethanesulfonyl fluoride was then added to the cell samples, followed by centrifugation at 14,000 g for 15 min at 4°C to extract total proteins. Protein concentrations were measured using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Jiangsu, China), and protein samples were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk/Tris-buffered saline–Tween for 2 h, and then incubated with the following primary antibodies: PKC, phospho-IκB, phospho-ERK, phospho-JNK, phospho-p38, IκB, ERK, JNK, p38, and GAPDH (1:500–1000 dilution) overnight at 4°C. The membranes were subsequently washed three times with Tris-buffered saline–Tween and incubated with secondary IRDye^®-conjugated goat anti-mouse or goat anti-rabbit antibodies (LI-COR Biosciences, Lincoln, NE, United States) at 1:8000 dilution for 50 min at room temperature. The membranes were washed a further three times, and the bands were scanned and their intensities measured using an Odyssey infrared imaging system (Gene Company, Beijing, China).

Frozen lung tissue (30 mg) was minced and homogenized in ice-cold lysis buffer containing Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich) and phenylmethanesulfonyl fluoride. The concentration of total protein was measured using a bicinchoninic acid assay kit according to the manufacturer’s instructions, with 30 μl protein per lane for separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer to a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat dried milk and then incubated with p38 MAPK, phospho-p38 MAPK, JNK MAPK, phospho-JNK MAPK, ERK MAPK, phospho-ERK MAPK, IκB, phospho-IκB, and GAPDH antibodies, respectively (1:1000 dilution) at 4°C overnight. After washing three times with TBST, IRDye-conjugated goat anti-mouse or goat anti-rabbit antibodies (1:5000 dilution, LI-COR Biosciences, Lincoln, NE, United States) were added at room temperature for 1 h. The intensities of the bands were then scanned and visualized using an Odyssey infrared imaging system (Gene Company, Beijing, China).