Western Blot Analysis of VEGF-A Expression

Western blot was conducted as previously described with modifications [24 (link)]. The cell lysate was separated under reducing conditions on 12% SDS–polyacrylamide gel and transferred onto polyvinylidene fluoride (PVDF) membranes. After blotting the gel, the PVDF membrane (Carl Roth GmbH, Karlsruhe, Germany) was blocked with 4% skim milk in Tris-buffered saline with 0.1% Tween for 1 h at room temperature. The blot was treated with the first antibodies, against beta-actin (Cell Signaling Technology, Leiden, Netherlands) or VEGF-A (A 20; Santa Cruz Biotechnology, Heidelberg, Germany), overnight at 4 °C in 2% skim milk in Tris buffered saline with 0.1% Tween. After washing, the blot was incubated with anti-rabbit immunoglobulin G (IgG), horseradish peroxide-linked antibody (Cell Signaling Technologies) in 2% skim milk in Tris-buffered saline with 0.1% Tween. Following the final washing, the blot was incubated with Immobilon chemiluminescence reagent (Millipore, Schwalbach, Germany), and the signal was detected with MF-ChemiBis 1.6 (Biostep, Jahnsdorf, Germany). The density of the bands was evaluated using Total laboratory software (Biostep), and the signal was normalized for β-actin.