Characterization of the VEGF-D binding domains in VEGFR-3. (A) Thermodynamic analysis of the Fc-tagged VEGFR-3D1-3 (domains 1-3) interactions with the VEGF-D (Cys117Ala) variants D_89-195, D_92-195, and D_100-195. The binding affinities (Kd) are indicated. ND indicates not determinable. (B) VEGF-D D_92-195 complexation with soluble, Fc-tagged VEGFR-2 domains 2 and 3 (R2D23), VEGFR-3 deletion mutants (R3D1-R3D123), and VEGFR-1-D1/VEGFR-3-D2 (R1/3D12) chimera. SDS-PAGE analysis of protein A pull-down assays is shown with Coomassie Blue staining. (C) Representative thermodynamic titrations of the VEGFR-3 deletion mutants (R3D2 and R3D12) and the VEGFR-1/VEGFR-3 (R1/3D12) chimera with VEGF-D D_92-195. (D) Summary of the VEGF-D D_92-195 binding experiments with Fc-tagged VEGFR-3 deletion mutants and the VEGFR-1/VEGFR-3 (R1/3D12) chimera. The enthalpy change (ΔH ± SD), entropy change (ΔS), binding affinity (K_d ± SD), and stoichiometry (n) of the thermodynamic binding experiments are shown. ND indicates not determinable.