Fig. 6. Combined effects of individual uPA domains leading to enhancement of cell migration and association of uPAR with vß5 integrin. (A,B) HEK-293/uPAR-25 cells were allowed to migrate towards (A) increasing concentrations of the indicated effectors for 3 hours or (B) 0.1 nM of the effectors for 15, 30, 60 and 120 minutes (B). In the combinations, a molar ratio of 1:1 was employed. When indicated, CPp or uPA 1-158 were preincubated with 100 ng of purified vß5 integrin in 0.2 ml of 0.1% BSA in DMEM for 1 hour at 37°C. In all cases, cells were assayed for directional migration in Boyden chambers as specified in the legend to Fig. 1. The results are expressed as mean ± s.d. of three independent experiments performed in triplicate. (C) Lysates (400 µg/sample) of HEK-293/uPAR-25 cells exposed for 60 minutes to the indicated peptides or diluents (NT), were immunoprecipitated with 5 µg/ml VNR147 anti-v integrin monoclonal antibody. 1 µg of anti-v integrin antibody was loaded as a control. The resulting proteins were analysed by western blotting using R4 anti-uPAR or polyclonal anti-v integrin antibodies.