FIGURE 3.

TM domain plays important role in proMMP-2 activation. A, schematic representation of mutant MT1-MMPs used. TM-NGFR, nerve growth factor receptor derived transmembrane domain. B, ProMMP-2 activation ability of these mutant MT1-MMPs were analyzed as in Fig. 1B. ProMMP-2 activation in the media was analyzed by zymography (upper panel), and cell lysates were analyzed for expression of the proteins by Western blotting using anti-FLAG M2 antibody (lower panel). The arrows in the Western blot (lower panel) indicate MT1-MMP mutants expressed. P, proMMP-2; A, active MMP-2. C, transfected cells were subjected to surface biotinylation as described under “Materials and Methods” and analyzed by Western blotting using anti-FLAG M2 antibody. The upper panel is biotinylated samples, and the bottom panel is whole cell lysates. The arrows in Western blot indicate MT1-MMP mutants expressed. D, in situ gelatin degradation assay was carried out as described under “Materials and Methods.”