Figure 3

Interaction strengths between Unc5b and Flrt paralogs are surprisingly heterogeneous. (a) Equilibrium binding analysis of Unc5b and three Flrt paralogs. Different concentrations of purified, monomeric Unc5b-Cd4d3+4-6H were injected over streptavidin-coated flow cells upon which biotinylated baits - Flrt1a (1018 RU), Flrt1b (984 RU), Flrt3 (1027 RU) - and control Cd4d3+4 were immobilized. The amount of bound Unc5b was calculated by subtracting responses in the control flow cells from those in the Flrt-immobilized cells once equilibrium had been reached. Equilibrium dissociation constants (K_Ds) were obtained by fitting a non-linear binding curve to the data. To facilitate comparison, the binding responses were normalized by using the predicted R_max from the fit to the data. (b) Kinetic analysis of the Unc5b-Flrt interactions. Off-rate constants (k_off) were calculated by globally fitting a first order decay curve to the dissociation phase of three concentrations of Unc5b; half-lives (t_1/2) were calculated as t_1/2 = ln2/k_off. Shown are the normalized, averaged values (error bars = ± 1 standard deviation, n = 3). On-rate constants (k_on) were calculated in the same way using an association model and were > 1 × 10⁵ M^-1s^-1 in all cases.