Specificity shift of uPA^{Y23N-R28N-R30H-R31W} from mouse to human uPAR. (A) The receptor-binding status of uPA in urine was tested by ligand blotting on PVDF membranes after SDS-PAGE of 0.5 μg purified mouse uPAR (m, lanes 1, 3, 5, 7, 9, 11, and 13) or human uPAR (h, lanes 2, 4, 6, 8, 10, 12, and 14). In lanes 1 to 4, the species selectivity of the uPAR-uPA interaction is demonstrated by incubating the blot with 1nM purified mouse pro-uPA (rmuPA, lanes 1-2) or human pro-uPA (rhuPA, lanes 3-4) spiked in urine from Plau^−/− mice. In lanes 5 to 10, pools of void urine from Plau^+/+ (n = 3; lanes 5-6), Plau^−/− (n = 3; lanes 7-8), and Plau^GFDhu/GFDhu (n = 7; lanes 9-10) mice were incubated with the uPAR blots. Bound uPA was detected with polyclonal rabbit anti-uPA antibodies, followed by swine anti–rabbit anti-IgG. In lanes 11-14, the specificities of these interactions were verified by incubating Plau^+/+ (lanes 11-12) and Plau^GFDhu/GFDhu (lanes 13-14) urine in the presence of 20nM recombinant human pro-uPA followed by a rabbit polyclonal antibody specific for mouse uPA. All urine samples were diluted 20-fold in PBS containing 0.05% (vol/vol) Tween 20 before incubation overnight at 4°C. The position of molecular weight markers (kDa) is indicated at left. (B) 4 adult Plau^+/+ (left column) and 4 Plau^GFDhu/GFDhu (right column) mice were injected intraperitoneally with 8 mg/kg PrAg-U2 and 10 mg/kg FP59. The health of the mice was determined by an investigator unaware of animal genotype. The mice were then humanely killed when moribund (P = .014; Fisher exact test).