Western Blot Analysis of VEGF

Lung tissues were homogenized in ice-cold buffer, sonicated, and centrifuged to remove cellular debris. 30 μg proteins were resolved and electroblotted to a polyvinylidene difluoride membrane (ImmobilonP, Millipore, Bedford, MA, USA). The membranes were incubated with antibody against VEGF (Santa Cruz Biotechnology) and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) and subsequently with horseradish peroxidase-conjugated goat antimouse IgG (Pierce Biotechnology, Rockford, IL, USA) after being blocked with 5% nonfat dry milk. We used SuperSignal Substrate from Pierce Biotechnology to detect protein bands. Densitometric analysis was performed using AIDA software to measure the intensity of VEGF and β-actin bands.