Fig. 1. Characterization of antibody 733 specificity. (A) Immunoblot analysis. Lysates of heparanase-transfected HEK-293 cells were resolved by SDS-PAGE and blots were probed with antibody 1453 (left panel), 733 (middle panel) and anti-heparanase monoclonal antibody purchased from Becton-Dickinson (BD, right panel). (B) Metabolic-labeling followed by immunoprecipitation. Heparanase-transfected CHO cells were pulsed for 20 minutes with [³⁵S]methionine (0) and then chased for the indicated time intervals in complete growth medium containing an excess of non-radioactive methionine. Equal volumes of cell lysate samples were subjected to immunoprecipitation (IP) with mAb 130 (top panel) or antibody 733 (bottom panel), as described in Materials and Methods. (C) Immunohistochemistry. Placenta sections (5 µm) were subjected to immunostaining with antibody 733, as described in Materials and Methods. Negative control, in which the primary antibody was omitted, is shown in the bottom panel. Original magnifications: top and bottom panels x20, middle panel x100.