HPLC binding data for UN2A and S100A1 in the presence and absence of Ca²⁺. (A) S100A1 binds UN2A in the presence of Ca²⁺. Purified UN2A (dark gray, dashed line) and S100A1 (light gray, dotted line) were incubated at pCa 3 and separated on TSKgel G2000SWxl HPLC column. The retention time of the UN2A/S100A1 complex (black, solid line) decreases with increasing Ca²⁺ concentration, suggesting a Ca²⁺-dependent binding interaction. (B) S100A1 does not bind UN2A in the absence of Ca²⁺. Purified UN2A and S100A1 were incubated at pCa 10 and separated on TSKgel G2000SWxl HPLC column. In the absence of Ca²⁺, the UN2A/S100A1 complex (dashed line) shows two separate peaks corresponding to UN2A (retention time = 9.875 min) and S100A1 (retention time = 11.005 min), indicating no binding interaction. (C) Effect of Ca²⁺ and S100A1 concentration on UN2A’s retention time. Plots depict the normalized retention time of UN2A at 20 µM relative to varying concentrations of S100A1 at pCa 3 (dashed line) and pCa 10 (solid line). The retention times were normalized to mitigate experimental variations. At pCa 3, a decrease in the retention time of UN2A suggests a complex formation at higher S100A1 concentrations. At pCa 10, the retention time of UN2A remains stable, indicating no complex formation with S100A1.