NRP-2 associates with VEGFR-2 and VEGFR-3 in a ligand-dependent manner in HEK cells. HEK cells were transfected with an empty vector (plasmid), NRP-2–Flag, VEGFR-2, HA–VEGFR-3 or cotransfected with NRP-2–Flag and either VEGFR-2 or VEGFR-3HA. Cells were either left unstimulated (A-B) or stimulated (C-D) with VEGF-A 10 ng/mL (lane A) or VEGF-C 100 ng/mL (lane C) for 10 minutes at 37°C. After immunoprecipitation with anti-Flag antibody, the receptors were immunoblotted with anti-Flag and anti–VEGFR-2 (A,C) or with anti-Flag and anti-HA (B,D). Bands were quantified and normalized to total amount of NRP-2 and then compared with unstimulated cells. The resulting fold increase of VEGFR-2/NRP-2 and VEGFR-3/NRP-2 association in response to VEGF-A and VEGF-C is indicated below the Western blots. Results are representative of 3 independent experiments with similar results.