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).