Immunoblot Analysis of Lung Protein Expression

Total protein was isolated from lung tissue homogenates. The lysate was centrifuged at 11,000 g for 1 min at 4°C. Supernatants were collected and the protein content was quantitated using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). Equal amounts of total protein were separated using 12% SDS polyacrylamide gels, and then transferred on to PVDF membranes (Millipore, USA). After blocking with 5% milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at 37°C, membranes were incubated with rabbit anti-actin monoclonal antibody (1:1000 dilution, ab179467, Abcam), rabbit anti-VEGF Receptor 1 monoclonal antibody (1:2000 dilution, ab32152, Abcam), or rabbit anti-PGF polyclonal antibody (1:1000 dilution, TA332424, OriGene Technologies, USA) at 37°C for 1 h. After washing with TBST three times, all membranes were incubated with goat anti-rabbit IgG H&L (HRP) preadsorbed secondary antibody (1:5000 dilution) (Abcam) at 37°C for 40 min. After washing with TBST three times, protein expression was visualized using Immobilon Western Chemilum HRP Substrate (Millipore). Actin served as an internal loading control. Relative protein expression was calculated using a method described previously (25 (link)).