VEGF Protein Interaction Profiling

Whole cell lysates from the cultured HEK293 cells and normal placental tissue (total 1 mg of protein) were incubated with VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. Following incubation, immunocomplexes were immunoprecipitated using protein A/G agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C with gentle rotation. The beads were washed three times with lysis buffer and boiled in 50 μl of 1X SDS sample buffer for 5 min at 95 °C. After centrifugation, the precipitated proteins were separated by SDS-PAGE and electrophoretically transferred onto an enhanced chemiluminescence (ECL) nitrocellulose membrane (GE Healthcare, London, UK). Western blot analysis was performed with the indicated primary antibodies followed by appropriate horseradish peroxidase–conjugated secondary antibodies using an ECL system (GE Healthcare, Buckinghamshire, UK), as described previously^{45 (link)}. Equal protein loading was confirmed by Ponceau S staining and sequential incubation of the membrane with anti-β-actin (Sigma-Aldrich) antibody.