Functional analysis of VEGFR-3 tyrosine mutants. (A) Schematic representation of the EGF–VEGFR-3 chimera. The large oval represents the extracellular EGFR domain. Tyrosine residues are indicated by circles. • represents the tyrosine residues that have been mutated in this study. Positions of the tyrosines are indicated by numbers. Deletion mutant is indicated by an arrowhead. (B) HUVECs expressing the chimeric E-R3 mutant receptors untreated or treated with 10 ng/mL EGF for 10 minutes. Immunoprecipitates with EGFR receptor were revealed with anti–VEGFR-3 C-terminal domain and antiphosphotyrosine-specific antibodies as indicated. (C) Quantitative analysis of 3 independent sprouting experiments measured by digitized image analysis of HUVECs embedded as spheroids in a collagen matrix treated with 10 ng/mL EGF and photographed 18 to 24 hours after induction. Quantification of the cumulative sprout length of 5 spheroids per experimental group. The results are expressed as the mean ± SD of 3 independent experiments.