Fig. 2. Identification of an NMR-friendly divalent cation.

(A) ITC of the interaction between αKG and Alkbh5 in the presence of Mn²⁺ (panel 1), Zn²⁺ (panel 2), Cd²⁺ (panel 3), and Mg²⁺ (panel 4) obtained at pH 6.0 and 25°C. Integrated heats of injection (black squares) and least-squares best-fit curves (red lines) derived from a simple one-site binding model are shown. The equilibrium dissociation constants and thermodynamic parameters are given in Table 1. (B) Pull-down assay on the interaction between Alkbh5 and a DNA oligonucleotide containing the m⁶A modification. Lanes 1, 3, 6, and 8 show results for the pull-downs acquired in the presence of Mn²⁺, Zn²⁺, Cd²⁺, and Mg²⁺, respectively, and in the absence of αKG. Lanes 2, 4, 7, and 9 show results for the pull-downs acquired in the presence of Mn²⁺, Zn²⁺, Cd²⁺, and Mg²⁺, respectively, and in the presence of αKG. Lane 5 contains a molecular weight marker.