Ca²⁺-dependent interaction of ID4 mRNA with hnRNP A2. In EMSA experiments using native PAGE, shifts to lower mobility reveal binding of radiolabeled RNAs to recombinant hnRNP A2. (A and B) Mobility shifts indicate that binding of ID4-chimeric α-tubulin mRNA to hnRNP A2 was dependent on Ca²⁺ levels. Maximal binding was observed at 500 nM Ca²⁺. Original data are shown on the left (A), and combined results from five experiments are shown in the diagram on the right (B). (C–H) Equilibrium binding constants were established in a series of experiments in which ID4-chimeric α-tubulin mRNA was titrated with increasing concentrations of hnRNP A2 at different concentrations of Ca²⁺. Original data are shown on the left (C, E, and G). The binding data were fitted to the Hill equation as previously described (Ryder et al., 2008; Chao et al., 2010; Muslimov et al., 2011) and were plotted in binding curves on the right (D, F, and H). Equilibrium dissociation constants were as follows: K_d = 280 nM at 100 nM Ca²⁺ (D), K_d = 200 pM at 500 nM Ca²⁺ (F), and K_d = 580 nM at 2 µM Ca²⁺ (H). Error bars indicate SEM.