Expression and regulation of VEGFRs in HUVECs. HUVECs were treated either with DPBS or VEGF analogs (10^–9 M; 7.5 minutes). Where indicated, HUVECs were pretreated for 15 minutes either with a VEGFR-2 inhibitor or PAFR antagonists prior to stimulation with VEGF analogs. Lysates were prepared and VEGFR-1 (A,E), VEGFR-2 (B,C,F), VEGFR-3 (D), or NRP-1 (G) were immunoprecipitated (IP) from 1 mg lysate. Following resolution on SDS-PAGE and transfer onto polyvinylidene difluoride (PVDF) membranes, immunoreactive bands were visualized by enhanced chemiluminescence (ECL), digitized using a 2-dimensional gel scanner, and quantified using Quantity One software (Bio-Rad). Results were normalized to those from DPBS-treated cells (upper bands). Membranes were subsequently stripped using Re-Blot Plus Strong stripping solution and the detection of VEGFR-1, VEGFR-2, or VEGFR-3 phosphorylation was performed with antiphosphotyrosine antibodies. Phosphorylation results were normalized to those from cells treated with VEGF analogs (lower bands).