Fig. 5. Processing of membrane-targeted heparanase is chloroquine sensitive. (A) Expression of membrane-targeted heparanase in stably transfected 293 and C6 cells. Control (M) and heparanase (Hepa) transfected cells were lysed and subjected to immunoblot analysis with anti-heparanase antibody 1453 (upper panel) or anti-actin antibodies (lower panel). 293 cells expressing the membrane-targeted heparanase gene construct were analyzed by FACS (B) with anti-heparanase monoclonal antibody (BD, upper panel) and anti-c-Myc epitope-tagged antibodies (lower panel), or stained by immunofluorescence (C) with anti-heparanase monoclonal antibodies (BD). Negative control, in which the primary antibody was omitted, is shown in the lower panel. Note heparanase accumulation at areas of cell-cell junctions in sparse cultures (C, top panel) and exclusive localization at the cell borders in confluent cells (C, middle panel). (D) Processing of membrane-targeted heparanase is chloroquine sensitive. 293 (left panel) and C6 (right panel) cells stably transfected with the membrane-targeted heparanase gene construct were left untreated (Con) or treated for 20 hours with 100 µM chloroquine (Chl). Total cell lysates were immunoblotted with anti-heparanase antibody 1453. Non-transfected cell lysates were included as control (M).