uPAR colocalizes with lipid rafts in MDA-MB-231 and ZR 751 cells. (A) and (B) MDA-MB-231 and ZR 751 cells grown in 8-well chamber slides were serum starved for 12-16 hrs, either left untreated or pretreated with MβCD, washed, labeled for the lipid raft marker GM1, fixed, permeabilized and blocked with 5% BSA for 1 hr. Then, cells were washed, labeled with anti-uPAR antibody for 1 hr at room temperature, washed, and stained for 1 hr with Alexa Fluor 488 (uPAR) and Alexa Fluor 594 (GM1) conjugated secondary antibodies at room temperature. Panels a to c: untreated cells; panels d to f: cells pretreated with 7.5 mM MβCD. The insets in panel's c and f show an enlarged view of the colocalization. Arrows indicate colocalization of uPAR with GM1 in MDA-MB-231 cells and uPAR and flotillin in ZR 751 cells before and after treatment with MβCD. Scale bars: 10 μM. (C) Inhibition of uPA activity by fibrin zymography. MDA-MB-231 and ZR 751 cells were left untreated or pretreated with different concentrations of MβCD, treated with ruPA (positive control) (10 ng/μl) washed and serum-free medium was added. After 24 hrs, conditioned medium was collected, and uPA activity was measured by fibrin zymography in MDA-MB-231 and ZR 751 cells. Densitometric analysis was done using the ImageJ software (**p < 0.001). (D) Western blot analysis of uPAR protein expression at 1 hr and 24 hr time points in cell lysates from MDA-MB-231 (D) and ZR 751 (E) cells either left untreated or pretreated with different concentrations of MβCD. Western blot analysis was performed with an antibody specific for uPAR. GAPDH was simultaneously immunodetected to verify equal loading of cell lysates. Densitometric analysis was done for uPAR expression (*p < 0.01). The result provided is a representative experiment repeated 3 times with concordant results.