Binding Affinity of AfEno1 with Regulators

The binding affinity of AfEno1 and factor H, C4BP, or plasminogen were determined by biolayer interferometry (Forte Bio, Menlo Park, CA) as previously described (19 (link), 34 (link)). For each concentration, Ni-NTA biosensors were hydrated in DPBS (0.001% gelatin) for 10 min; then recombinant AfEno1 was bound to biosensors for 120 s. After washing the sensor (30 s) with DPBS (0.001% gelatin), factor H, C4BP, or plasminogen at various concentrations were added as analytes. For each concentration, the association of the complexes was followed for 250 s. After washing the sensor, the dissociation of the complexes was followed for another 250 s. All interactions were performed at RT. As a non-binding control, heat inactivated regulators were used. The data were obtained after subtracting the buffer blank using a 1:1 model of interaction. Graphs were plotted in Graph Pad Prism 5.