Plasminogen binds to the C-terminal peptide of Plg-R_KT. The peptide, CEQSKLFSDK, corresponding to the amino terminus of Plg-R_KT was coupled to BSA and coated onto wells of microtiter plates. Either Glu-plasminogen (A) or Lys-plasminogen (B) or t-PA (C) was then incubated with the wells, followed by antiplasminogen mAb⁵⁴  (A-B) or anti–t-PA mAb (C) and detection with HRP-conjugated goat anti–mouse IgG (●) as described in “Plasminogen and t-PA binding assay.” The binding was specific because it was blocked in the presence of 0.2M EACA (△), consistent with the ability of EACA to inhibit plasminogen binding to differentiated Hoxa9-ER4 cells. In additional controls, nonspecific binding to either BSA (▲), or to the reverse peptide (○) was < 10% of binding to CEQSKLFSDK. (At high input concentrations of t-PA, nonspecific binding increased, but was < 10% of binding to CEQSKLFSDK at the concentration required for 50% saturation [3.2nM]). In controls for the detection method, optical density at 490 nm (OD₄₉₀) values obtained using an isotype control antibody or in the absence of added plasminogen or t-PA were < 5% of the values for plasminogen or t-PA binding to immobilized CEQSKLFSDK. (D-E) Biotinylated Glu-plasminogen (25nM) was incubated with immobilized CEQSKLFSDK in the presence of increasing concentrations of (D) the C-terminal peptide, CEQSKLFSDK (●) or a mutated C-terminal peptide with K147 substituted with alanine, CEQSKLFSDA (○) or (E) anti–Plg-R_KT mAb 35B10 (●) or isotype control (○). Biotinylated Glu-plasminogen binding was detected with HRP-streptavidin and was 96% inhibited in the presence of 0.2M EACA (not shown). Data are mean ± SEM; n = 3, for each determination.