Western blot

In snap frozen bulk lung tissue from the middle lobe of the right lung, total endothelial NOS (eNOS) and phosphorylated eNOS (Ser1177 site) were determined by SDS‐PAGE. Proteins were transferred to nitrocellulose membranes and blots were probed with primary anti‐phospho‐eNOS Ser1177 (1:1000, purified monoclonal rabbit anti‐human eNOS, CST9570, Cell Signaling Technology Inc., Danvers, MA, USA), anti‐eNOS (1:500, purified monoclonal mouse anti‐human eNOS, 610297, Transduction Laboratory, BD Biosciences, San Jose, CA, USA), and anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; 1:1000, 14C10, Cell Signaling Technology). All blots were analysed using the Odyssey CLX imaging system (LI‐COR Biotechnology, Lincoln, NE, USA). We performed Rout's test on the data, which identified one outlier in the NOI group.