Neutrophil gelatinase-associated lipocalin as a potential novel biomarker for ventilator-associated lung injury

Xiao, Rui; Chen, Rongchang

Western blotting. Lung tissue protein was extracted using ice-cold RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China) according to the manufacturer's protocols. Protein concentration was determined using the Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). Total proteins (30 µg) were separated by 8-12% SDS-PAGE at 120 V, and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) at 250 mA for 90 min via wet transfer. Following blocking non-specific binding sites with 5% non-fat milk in Tris-buffered saline (TBS) with Tween-20 (20 mM/l TBS pH 7.5, 500 mM/l NaCl and 0.1% Tween-20) for 1 h, the blots were probed with mouse monoclonal anti-β-actin (sc-47778; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-NGAL (MAB1857; 1:1,000; R&D Systems, Inc., Minneapolis, MN, USA) primary antibodies at 37˚C for 1 h. Bands were detected using an anti-mouse horseradish peroxidase-conjugated secondary antibody (7076; 1:2,000; Cell Signaling Technology, Inc.) at 37˚C for 40 min followed by treatment with SuperSignal ® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.) and X-ray film exposure (Kodak, Rochester, NY, USA). The film was scanned, and densitometric analysis was performed using QuantityOne software version 4.6.2 (Bio-Rad Laboratories, Inc.). Squares of equal size were drawn around each band to measure the density, and the value was adjusted based on the density of the background near that band. Densitometric analysis was repeated three times. Results were expressed as a ratio of the target protein compared with the reference protein. The ratio of the target protein from the control group was arbitrarily set to 1.