Gremlin binds VEGFR2. (A) sVEGFR2_D1-7 (Calbiochem) was immobilized at approximately .083 pmol/mm² to a CM5 sensorchip (BIAcore) that was previously activated with a mixture of 0.2M N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 0.05M N-hydroxysuccinimide (35 μL; flow rate: 10 μL/min). Increasing concentrations of gremlin were injected in HBS-EP buffer (BIAcore) for 4 minutes (sample volume: 40 μL; flow rate: 5 μL/min; dissociation time: 4 minutes). The response (in response units) was recorded as a function of time. An overlay plot is shown of all sensorgrams after subtraction of their respective control sensorgrams. Binding parameters, calculated by the nonlinear-curve–fitting software package BIAevaluation 3.2 (BIAcore Inc) applied to all sensorgrams simultaneously using a single-site model with drifting baseline, indicate that gremlin/VEGFR2 interaction occurs with Kd = 47 ± 15nM. Under the same experimental conditions, VEGF-A/VEGFR2 interaction occurs with Kd = 3 ± 1nM. (B) Gremlin (25nM) was injected over the sVEGFR2-coated sensor chip in the absence or in the presence of soluble sVEGFR1-Fc, sVEGFR2-Fc, sVEGFR3-Fc, or sFGFR1-Fc (all at 314nM). Binding data were plotted as percentage of maximal bound analyte (recorded at the end of injection) and represent the mean of 2-3 independent experiments. (C) Ninety-six–well plates coated with 100 μL of 250 ng/mL sVEGFR1-Fc or sVEGFR2-Fc were incubated with VEGF-A (20 ng/mL dissolved in phosphate-buffered saline containing 0.1% BSA, 5.0mM (ethylenedinitrilo)tetraacetic acid, 0.004% Tween 20 in presence of different competitors and incubated for 1 hour at 37°C followed by 1-hour incubation at room temperature. Bound VEGF-A was detected with an anti–human VEGF monoclonal antibody (R&D Systems). Gremlin competes with VEGF-A for the binding to immobilized sVEGFR2-Fc (□) in a competitive ELISA for which VEGF-E (○) and FGF2 (▵) were used as positive and negative controls, respectively. At variance, gremlin did not compete with VEGF-A for the binding to immobilized sVEGFR1-Fc (■), whereas placenta growth factor (PGIF) (▾) was fully effective. (D) HUVECs were incubated with 5.0nM gremlin conjugated with the bifunctional photoactivable biotin-label transfer cross-linker Sulfo-SBED Biotin Label transfer reagent (Pierce) (sulfo-gremlin) in the absence or in the presence of a molar excess of unlabeled gremlin or VEGF-A. After ultraviolet irradiation, cell lysates (1.0 mg of protein) were immunoprecipitated with anti-VEGFR2 antibody (Santa Cruz Biotechnology), separated on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel under reducing conditions and probed with streptavidin–horseradish peroxidase to visualize the biotin–VEGFR2 complex. Uniform loading of the gel was confirmed by probing the membrane with the anti-VEGFR2 antibody.