Localization of uPA, uPAR and β1-integrin to caveolae-enriched fractions of SUM149 cells. Isolation of caveolae from SUM149 cells grown in (a) 2D or in (b) 3D culture was performed using a non-detergent method as described in Materials and Methods. Equal-volume aliquots of fractions 3-11 were analyzed by SDS-PAGE and immunoblotted with antibodies against uPA, uPAR, β1-integrin, or caveolin-1. Merged DIC and fluorescent images are representative images of SUM149 (c) 2D or (d) 3D cultures immunostained for caveolin-1 (green) and uPA (red) or uPAR (red). Colocalization of the two proteins appears yellow (arrows). Confocal microscopy was performed as described in Materials and Methods. Bar, 10 μm for 2D cultures; 20 μm for 3D cultures. uPA, urokinase-type plasminogen activator; uPAR, urokinase receptor.