NP-40 (P0013F) was purchased from Beyotime for the extraction of total protein in airway tissues. 25 mg of airway tissues stored at −80°C were lysed with 300 µL of NP-40 solution at 4°C. After the tissues were centrifuged for 30 min at 4°C and 14,000 × g, the total protein was obtained. Then, a BCA kit (P0009, Beyotime) was further applied for the determination of protein concentration. After the protein concentration in each group was determined, 25 µg of protein in each group and 4 µL of marker (PR1910, Solarbio) were transferred into the designed lane of the 10% SDS-PAGE gels (P0052A, Beyotime). Then, the protein swam vertically down inside the gels under 100 volt for 120 min, and then transferred to the surface of 0.45 μm membranes (ISEQ00010, Millipore, MA, USA). The pore without target proteins on the membranes was blocked with 5% non-fat milk, and then the membranes were incubated with relative primary antibodies at 4°C overnight for target protein detection. The relative primary antibodies were Wnt5a (1:1500, 42kD, ab229200, Abcam), β-catenin (1:1000, 86kD, ab32572, Abcam), VEGFA (1:2500, 27kD, ab52917, Abcam), TGF-β1 (1:1000, 44kD, ab92486, Abcam), Fibronectin (1:1500, 262kD, ab2413, Abcam), MMP-9 (1:2500, 92kD, ab38898, Abcam), and GAPDH (1:5000, 36kD, ab181602, Abcam). The second day, the rabbit secondary antibody (1:20,000, ab205718, Abcam) was applied to further incubate with all the membranes for 2 h at room temperature. Finally, each membrane was covered with 200 μL of detection solution (P0019, Beyotime), and the image signal was detected using Bio-Rad ChemiDoc™ XRS+ System and the Image Lab 3.0 Software (Bio-Rad, CA, USA).

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