Whole membrane preparation
Lower and upper regions of AGM lungs were dissected by visual differentiation. Whole membranes were prepared from individual AGM lungs as previously described (Miksys & Tyndale, 2002). Briefly, lung tissues were homogenized in 0.1 mM EDTA, 0.1 mM DDT, 100 mM Tris, and 0.32 M sucrose (pH 7.4), then centrifuged at 3000 × g for 10 minutes at 4°C. The supernatant was collected, the pellet was resuspended, vortexed and centrifuged again at 3000 × g for 10 minutes. The two supernatants were combined and centrifuged at 110,000 × g for 60 minutes at 4°C. The resulting pellet was resuspended in storage solution consisting of 0.1 mM EDTA, 0.1 mM DDT, 100 mM Tris with 1.15% KCl and 20% glycerol. The protein contents of the lung membranes were measured using a Bradford Protein Assay kit (Bio-Rad; Burlington, ON, Canada). Pooled Control group lung membranes for lower and upper lung were created by combining equal amounts of lung membrane protein from each control AGM in Study 1 (n=6) and separately for Study 2 (n=10).