The interactions between the peptides and the compounds were analyzed with the Biacore T200 system (GE Healthcare, Uppsala, Sweden) at 25°C. The recombinant eboIZN39IQ (N39) peptide was immobilized on a sensor chip (CM5) with the Amine Coupling Kit (GE Healthcare, Buckinghamshire, UK). The final levels of immobilized eboIZN39IQ (N39) were typically approximately 3000 response units (RU). The eboC24 (C24) peptide was conjugated to DIBO-biotin (Invitrogen) by click chemistry and then immobilized on an SA sensor chip, with final levels of approximately 600 RU. Various concentrations of the compounds were subsequently injected as analytes, and PBS-P [10 mM phosphate buffer containing 2.7 mM KCl, 137 mM NaCl, and 0.05% surfactant P20 (pH 4.5)] was used as the running buffer. For the binding studies, appropriate concentrations of the analytes were added to the running buffer at a flow rate of 30 μl/min, a contact time of 120 s, and a dissociation time of 60 s, and the chip platforms were washed with running buffer and 50% DMSO. The data were analyzed with Biacore evaluation software (T200 version 1.0), and the curve was fitted with a 1:1 binding model.

The eboIZN39IQ (N39) peptide was immobilized on a CM5 sensor chip at approximately 3000 RU to explore whether Y11-eboC24 binding had any effect on the interactions between the eboC24 and eboIZN39IQ (N39) peptides in the SPR experiments. EboC24 concentrations of 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, and 0 μM were passed through the chip at a flow rate of 10 μl/min, a contact time of 60 s, and a dissociation time of 600 s in the absence or presence of Y11 or other compounds. The N39 peptide-SA chip was regenerated with glycine (pH 2.5). The data were analyzed with Biacore evaluation software (T200 Version 1.0), and the curve was fitted with a 1:1 binding model.

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