Figure 4.

Binding Characteristics of VEGF₁₆₅a Binding to NanoLuc-NRP1

(A) Increasing concentrations of VEGF₁₆₅a-TMR were added to HEK293T cells stably expressing N-terminal NanoLuc-NRP1 in the presence and absence of 100 nM unlabeled VEGF₁₆₅a to determine non-specific binding, and cells were incubated for 60 min at 37°C. Raw BRET ratios are expressed as mean ± SEM from 5 independent experiments.

(B) Time course of VEGF₁₆₅a-TMR binding to NanoLuc-NRP1. BRET ratios were baseline corrected to vehicle, curves were fitted to a simple exponential association model, and data are shown as mean ± SEM from 5 independent experiments.

(C) Inhibition of the binding of VEGF₁₆₅a-TMR (0.5, 1, 2, 3, and 5 nM) to NanoLuc-NRP1 by increasing concentrations of unlabeled VEGF₁₆₅a added simultaneously and incubated for 60 min at 37°C. Raw BRET ratios from 5 independent displacement experiments using duplicate wells are shown as mean ± SEM with bars representing vehicle (white) or VEGF₁₆₅a-TMR only.

(D) Linear regression analysis (R² = 0.95; p < 0.005) of the relationship between IC₅₀ values determined in (C) and VEGF₁₆₅a-TMR concentration. The y intercept provides an estimate for the K_i of competing VEGF₁₆₅a (0.10 nM), while the slope (0.09) represents the ratio K_i/K_D thus yielding an estimated K_D = 1.11 nM for VEGF₁₆₅a-TMR at NanoLuc-NRP1.