Because uPAR colocalized with αvβ3, next we determined whether they are physically associated. Fibroblast surface proteins were labeled with membrane impermeable biotin, the cells were solubilized, and the lysate immunoprecipitated with antibodies to β3, uPAR, or α5β1. The coimmunoprecipitated surface proteins were detected with streptavidin-HRP, and the results are shown in
Figure 7E . An antibody to β3 (lane 1) immunoprecipitated αvβ3 and a complex banding pattern of surface proteins. Immunoprecipitation with an antibody to uPAR revealed a similar pattern of biotinylated proteins. uPAR is a glycosylated protein, detected as bands between 50 to 65 kDa.
24 Other molecular weight uPAR-containing species are detectable also, such as uPAR bound to uPA or to the uPA inhibitor PAI-1/2.
25 This uPAR banding pattern was corroborated independently by Western blot detection
(Fig. 7F) , using a different antibody from the one used for immunoprecipitation. Thus, the similar banding patterns in both the β3 and uPAR immunoprecipitation suggest that these proteins are physically associated in the plasma membrane. However, it was surprising that bands representing αvβ3 were not observed (
Fig. 7E , lane 2), whereas two other high molecular weight bands were identified (
Fig. 7E , lane 2, stars). It was conceivable that the anti-uPAR antibody did not recognize uPAR that was bound to β3. In such a β3-uPAR complex, the anti-uPAR recognition site may be sequestered. To test this, we performed sequential immunoprecipitation. Lysates were incubated with anti-uPAR antibody to remove uPAR that readily bound to anti-uPAR, and then, the remaining supernatant was immunoprecipitated with anti-β3 in hopes that this would bring down the β3-uPAR complex. A 5-minute chemiluminescence exposure revealed a fraction of uPAR that coimmunoprecipitated with β3 even after uPAR was removed from the lysate with anti-uPAR antibody (
Fig. 7E , lane 3). We therefore conclude that the uPAR antibody (lane 2) did not immunoprecipitate uPAR that was bound to β3, whereas the β3 antibody recognized β3 that was bound to uPAR (
Fig. 7E , lanes 1 and 3). The sensitivity of the biotin-streptavidin system was necessary to demonstrate that uPAR immunoprecipitated with integrin, and the final identification was by characteristic molecular weight. This is in contrast to reports of cancer cells in which both uPAR and integrins are pathologically overexpressed
8 and in which the immunoprecipitated proteins can be identified by Western blot. Finally, as predicted by the lack of colocalization of α5β1 and uPA, anti-α5β1 did not immunoprecipitate uPAR (
Fig 7E , lane 4).