Fig. 7

ITC analysis of Mg²⁺ (A, D), Ca²⁺ (B, E) and Zn²⁺ (C, F) binding to ggNesfatin-3 (A–C) and hsNesfatin-3 (D–F). The top panels present the thermograms corrected for the baseline, and the color lines show the control titrations. The middle panels represent the binding isotherm with the fitted models. Residuals are shown at the bottom of each panel. For the titration of Zn²⁺ to ggNesfatin-3 (C), the sum of the single site (light red dashed line) and sequential two site (red dashed line) models is presented as a dark red solid line. In the case of Zn²⁺ titration on hsNesfatin-3 (F), the sum of the single site (light red dashed line) and linear blank (red dashed line) models is presented as a dark red solid line. All experiments were performed in 20 mM Hepes (pH 7.5), 150 mM NaCl at 25 °C