Knockdown of ECSCR in HUVECs results in reduced VEGF-induced phosphorylation of VEGF receptor 2/KDR and downstream PLCβ3, but not tyrosine phosphorylation of VEGF receptor 1/FLT1. (A) HUVECs transfected with the indicated siRNAs were serum starved overnight and stimulated with 25 ng/mL VEGF for the indicated periods. Total lysates were blotted using the indicated phosphoepitope-specific antisera. ECSCR knockdown cells show strongly reduced tyrosine phosphorylation of KDR on multiple epitopes. (B) Total lysates as described in panel A were blotted using anti-PLCβ3 phosphoepitopes. Activating phosphorylation of serines 537 and 1105 was reduced in ECSCR knockdown cells. (C) FLT1 was immune precipitated from unstimulated and VEGF-stimulated cells (5 minutes), and immune precipitates were blotted for phosphotyrosine. Tyrosine-phosphorylated FLT1 was present at similar levels in VEGF-stimulated control lacZ and ECSCR knockdown cells. Results were observed in at least 2 independent experiments.