MMP-9 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 different concentrations of MβCD, washed, labeled for the lipid raft marker GM1 using vibrant lipid raft labeling kit, fixed, permeabilized and blocked with 5% BSA for 1 hr. Then, cells were washed, labeled with anti-MMP-9 antibody for 1 hr at room temperature, washed and stained for 1 hr with Alexa Fluor 488 (MMP-9) conjugated secondary antibody 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 to f showed an enlarged view of the colocalization. Arrows indicate colocalization of MMP-9 with GM1 in MDA-MB-231 cells and with flotillin in ZR 751 cells. Scale bars: 10 μM. (C) Inhibition of MMP-9 activity as shown by gelatin zymography in MDA-MB-231 and ZR 751 cells left untreated, pretreated with different concentrations of MBCD or treated with rMMP-9 (2.5 ng/μl). Densitometric analysis was done using the ImageJ software (**p < 0.001). (D) Western blot analysis of MMP-9 protein expression at 1 hr and 24 hr time points in cell lysates from MDA-MB-231 (D) and ZR 751 (E) cells left untreated or pretreated with different concentrations of MβCD. Western blot analysis was performed with an antibody specific for MMP-9. GAPDH was simultaneously immunodetected to verify equal loading of the lysates. Densitometric analysis was done for MMP-9 expression (*p < 0.01, **p < 0.001). The result provided is a representative experiment repeated three times with concordant results.