Western Blot Analysis of Lung Tissue Proteins

As we described previously^{18 (link),19 (link)}, the left lung tissues (50 ug) were homogenized in RIPA buffer (catalogue number: 89900; Thermo Scientific, USA) containing phosphatase inhibitor cocktail (catalogue number: 04906845001; Roche Applied Science, USA) and protease inhibitor cocktail (catalogue number: P2714; Sigma, USA). Homogenates were centrifuged at 4 °C (13,000 rpm, 20 min). The supernatant was absorbed as the total protein. The protein concentration was measured by BCA protein assay according to the manufacturer’s instructions (catalogue number: 23227; Pierce Biotechnology, USA). Twenty microgram proteins each lane were loaded on a polyacrylamide gel and transferred onto polyvinylidene difluoride membrane. Then the proteins in membrane were incubated with the primary antibodies at 4 °C: anti-AQP1 antibody (1:1000, catalogue number: ab168387; Abcam, USA), anti-ICAM-1 antibody (1:1000, catalogue number: ab206398; Abcam, USA), anti-ZO-1 antibody (1:1000, catalogue number: ab190085; Abcam, USA), anti-MMP2 antibody(1:1000, catalogue number: ab37150; Abcam, USA), anti-MMP9 antibody(1:1000, catalogue number: ab38898; Abcam, USA), anti-β-Actin antibody (1:5000, catalogue number: ab50591; Abcam, USA). The protein bands were developed by enhanced chemiluminescence (Pierce, USA). The intensities of AQP1, ICAM-1, ZO-1, MMP2, MMP9 proteins were normalized by those of β-Actin.