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Fig. 2. Interaction between uPA, VN, suPAR and PN-1 in the solution phase. PN-1, VN, suPAR, uPA, PAI-1 and ATF (4 µg/ml each) were incubated in different combinations, as indicated, in TBS with 1% (w/v) BSA for 2 hours at 22°C. Thereafter, immunoprecipitation was performed with control mAb (lanes 1 and 8), anti-VN, 13H1 (lanes 2, 6 and 7), anti-uPAR, R4 (lane 5), anti-uPA, 4D1E8 (lane 4) or anti-PN-1, 4B3 (lanes 3 and 9) all at 4 µg/ml. The immune complexes were captured by adding protein A/G Sepharose and the immunoprecipitates were analyzed by western blotting with a rabbit polyclonal anti-uPAR followed by densitometric analysis. Similar results were obtained in three independent experiments.
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Vß3 (5 µg/ml)-coated wells (hatched bars) in the absence or presence of uPA (100 nM), PN-1 (100 nM), PAI-1 (100 nM) or a combination of uPA and PN-1. VN binding was detected with the mAb 13H1. (D) Binding of uPA (20 nM) to VN-coated wells (5 µg/ml) was tested in the absence (control) or presence of either PN-1 (100 nM), suPAR (50 nM) or a combination of the two as indicated. uPA binding was detected with the mAb 4D1E8. (E) Binding of VN (2 µg/ml) to uPA-coated wells (5 µg/ml) was tested in the absence (control) or presence of either PN-1 (100 nM), suPAR (50 nM) or a combination of the two as indicated. VN binding was detected with the mAb VN-7. (F) Binding of VN (1 µg/ml) to suPAR-coated wells (5 µg/ml) was measured in the absence (control) or presence of PN-1 (200 nM) or PAI-1 (100 nM), without any further addition (dotted bars) or together with enzymatically active uPA (20 nM; hatched bars) or enzymatically inactive Gly158-ScuPA (20 nM; chequered bars), respectively. VN binding was detected with the mAb 13H1. (G) Binding of uPA (20 nM) to VN-coated wells (5 µg/ml) in the presence of suPAR (50 nM) and increasing concentrations of PN-1 was tested in the absence (open square) or presence of thrombin (20 nM) (solid square) as indicated. Binding was detected with the mAb 4D1E8. (H) At the end of the incubation period in the above experiment the supernatants were removed and then analyzed for proteolytic activity using the chromogenic substrate S-2444, which is specific for uPA but not for thrombin. Results from a typical experiment are shown (mean absorbance ± s.e.m. of triplicate wells), and similar data were obtained in three separate experiments. Absence of error bars indicates that the s.e.m. is smaller than the size of the symbol.
