Western Blot Protein Analysis Protocol

Cells were lysed in PRO-PRE-Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Protein concentrations were determined using a Bradford assay kit (BIO-RAD, Hercules, CA, USA). Cell lysates (50 μg of total protein) were separated in 8% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-ECL nitrocellulose filter paper (Amersham Biosciences, Buckinghamshire, UK). Membranes were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. Protein bands were probed with VEGFR2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution; phospho-VEGFR2 (Tyr951, Tyr1175), total-ERK (Thr202/Tyr204), phosphor-ERK (Thr202/Tyr204), total-AKT, phospho-AKT total-p38, phospho-p38 (Cell Signaling, USA) at 1:1000 dilutions; β-actin antibody at a 1:4000 dilution (Santa Cruz Biotechnology, Santa Cruz, USA) and then labeled with horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare, Piscataway, USA). Bands were visualized by enhanced chemiluminescence using an ECL kit (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s protocol, as described previously^{23 (link)}.