Fig. 3. Heparanase uptake, processing and localization in human MDA-435 breast carcinoma and U87 glioma cells. MDA-435 cells were left untreated (Con, top row) or incubated with the 65 kDa heparanase precursor (5 µg/ml) for 5 minutes (second row), 1 hour (third row) or 3 hours (fourth row) and stained with monoclonal anti-heparanase antibody (BD, left column, red) or with antibody 733 (middle column, green). Merged images are shown in the right column. U87 cells were incubated with heparanase for 3 hours and similarly stained (fifth row). Original magnification: x100. MDA-435 cells were also stained with monoclonal anti-cathepsin D antibody, or with antibody 733. Merged image is shown in the right panel (bottom row). (Inset: top right) Heparanase uptake and processing. MDA-435 (upper panel) and U87 glioma cells (second panel) were left untreated (0) or incubated with the 65 kDa heparanase precursor. At the indicated time points, cells were washed and total cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibody 1453 (first and second panels), or with anti-actin antibody (third panel).