*2.7. Western Blot Analysis*

Mouse lung tissue extracts and A549 cell lysates were prepared in 1 mM Tris-HCl (pH 6.8) lysis bu ffer containing 10% sodium dodecyl sulfate (SDS), 1% glycerophosphate, 0.1 mM Na3VO4, 0.5 mM NaF, and a protease inhibitor cocktail. Tissue extracts and cell lysates containing equal amounts of proteins were electrophoresed on 8%–15% SDS-PAGE and transferred onto a nitrocellulose membrane. Blocking a nonspecific binding was performed using either 3% fatty acid-free BSA or 5% non-fat dry skim milk for 3 h. The membrane was incubated overnight at 4 ◦C with a specific primary antibody of COX-2, iNOS or ICAM-1, bcl-2, bax, cleaved caspases, MMP-12, or IκB. The membrane was then applied to a secondary antibody conjugated to HRP for 1 h. Following triple washing, the target proteins were determined using the Immobilon Western Chemiluminescent HRP substrate (Millipore Corp., Billerica, MA, USA) and the Agfa medical X-ray film blue (Agfa HealthCare NV, Mortsel, Belgium). Incubation with β-actin antibody was conducted for the comparative control.
