Lung tissue was obtained from 3 mice per group at time points after irradiation as indicated. One hundred milligrams of lung tissue was homogenized in 1 mL RIPA buffer containing protease inhibitors. Soluble proteins were separated from insoluble material by centrifugation (16,000g, 10 min), and the total protein count of the resulting supernatant was determined using the Bradford method (Bio-Rad). The supernatant was then subjected to ELISA to determine the concentrations of IL1-β, IL-4, TNF-α, EGF, TGF-β, IL-6 (mouse DuoSet ELISA, R&D Systems®, Minneapolis, MN) and TGF-α (human Quantikine ELISA, R&D Systems).
NIH-3T3 cells were incubated overnight in DMEM containing 1% FBS and 0.1% β-mercaptoethanol, followed by treatment with TGF-α (0–10 ng/ml). Culture supernatants were collected at 1, 24 and 72 h after TGF-α addition. Supernatants were subjected to ELISA to determine the concentration of TGF-β (mouse DuoSet ELISA, R&D Systems).
NIH-3T3 cells were incubated overnight in DMEM containing 1% FBS and 0.1% β-mercaptoethanol, followed by treatment with TGF-α (0–10 ng/ml). Culture supernatants were collected at 1, 24 and 72 h after TGF-α addition. Supernatants were subjected to ELISA to determine the concentration of TGF-β (mouse DuoSet ELISA, R&D Systems).