2D Proteomic Analysis of NHBE Cells

NHBE cells were harvested via centrifugation and then disrupted with a lysis buffer containing 5 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% Triton X-100, and 2 mM PMSF. The cell lysate was centrifuged at 12,000× g for 30 min, and the supernatant fraction was collected. The protein concentration was determined using a commercial BCA assay kit (Thermo, Chicago, IL, USA), and the samples were stored at −70 °C until use. Immobiline DryStrips (Amersham Biosciences, Piscataway, NJ, USA) were used for isoelectric focusing, which was carried out with 1 mg of the protein on an IPGphor system (Amersham Biosciences). After IEF separation, they were separated in the second dimension using SDS-PAGE. For the image analysis, they were visualized using the Coomassie Brilliant blue G-250 staining method. Next, the 2D spot intensity was calculated by integrating the optical density over the spot area. The values were normalized and then exported to the statistical analysis software. The experiments were performed at least two times [26 (link)].