Figure 8. Chemico-protein interactions analysis and competitive binding assay using Mg^2 +.

(A) Interactions between α-enolase (ENO1) and divalent cations commonly found in human urine (Ca²⁺, Mg²⁺, Mn²⁺, Zn²⁺ and Cu²⁺ ) were analyzed by using STITCH tool (version 4.0). (B,C) MDCK monolayers were pretreated with equal volume of MEM (blank control), Mg²⁺-free TBS (preservative or background control), or 0.1 M MgCl₂ in Mg²⁺-free TBS for 30 min prior to cell-crystal adhesion assay. Moreover, the divalent cation-binding sites on α-enolase were recovered by further incubating the cells with 5 mM EDTA in Mg²⁺-free TBS for 15 min prior to cell-crystal adhesion assay. Original magnification power was 400X. Each bar represents mean ± SEM of the data obtained from 3 independent experiments. *p < 0.05 vs. MEM, #p < 0.05 vs. TBS; ^†p < 0.05 0.1 M MgCl₂/TBS.