2.10. Lung Western Blotting Analysis

Western blot was performed using the protocol modified from Caperuto et al. in 2008 [27]. 50 μg of protein was loaded on an SDS-PAGE gel (10% bis-acrylamide) and then electrotransferred from the gel to a nitrocellulose membrane (10 minutes at 25 V constant) in a semidry transfer (Trans-Blot Turbo, Bio-Rad). Nonspecific binding protein in the nitrocellulose membrane was blocked with 5% milk/TBST and then incubated with anti-phospho-p38 MAPK (1 : 2,000, mouse IgG1, 9216), anti-p38 MAPK (1 : 1,000, rabbit IgG, 8690), anti-phospho-p44/p42 MAPK (ERK1/2) (1 : 2,000, rabbit IgG, 4370), anti-p44/p42 MAPK (1 : 2,000, mouse IgG1, 4696), anti-phospho-SAPK/JNK (1 : 2,000, mouse IgG1, 9255), anti-SAPK/JNK (1 : 1,000, rabbit, 9252), anti-phospho-STAT-3 (1 : 1,000, rabbit, 9131), anti-STAT-3 (1 : 1,000, mouse IgG2a, 9139), anti-SOCS-3 (1 : 1,000, rabbit, 2923) (Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (1 : 5000, mouse, A5316) (Sigma-Aldrich) diluted in blocking buffer for 4°C overnight. Bound antibodies were detected with horseradish peroxidase-conjugated (HRP-conjugated) anti-mouse IgG or anti-rabbit IgG (1 : 10,000, 7076 and 7074) (Cell Signaling Technology, Danvers, MA, USA) and visualized using UVItec (UVITEC Cambridge, England, United Kingdom) by chemiluminescence. The image program UVIband (UVITEC Cambridge) was used to quantify the intensities of the bands. The expression of all proteins was normalized to the constitutive β-actin protein, and all phosphorylated protein expression was normalized to its specific total protein. All the results were expressed as percentage related to the control (control group).