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 (KDs) were obtained by fitting a non-linear binding curve to the data. To facilitate comparison, the binding responses were normalized by using the predicted Rmax from the fit to the data. (b) Kinetic analysis of the Unc5b-Flrt interactions. Off-rate constants (koff) were calculated by globally fitting a first order decay curve to the dissociation phase of three concentrations of Unc5b; half-lives (t1/2) were calculated as t1/2 = ln2/koff. Shown are the normalized, averaged values (error bars = ± 1 standard deviation, n = 3). On-rate constants (kon) were calculated in the same way using an association model and were > 1 × 105 M-1s-1 in all cases.