Fig. 3. 1-PDX blocks gelatin degradation and proMT1-MMP processing in MT1-MMP-transfected A375 melanoma cells. (A) A375 cells transfected with MT1-MMP and pcDNA 3.0 (MT1) or MT1-MMP and FLAG-tagged 1-PDX (MT1/1-PDX) were cultured on surfaces coated with rhodamine-conjugated gelatin. After 16 hours of incubation, cells were fixed and labeled with specific primary antibodies: polyclonal immunopurified anti-MT1-MMP antibody MMR2 (MT1-MMP) and monoclonal anti-FLAG antibody M2 (1-PDX). Degradation of the underlying fluorescent gelatin by the MT1-MMP/pcDNA3.0 and MT1-MMP/1-PDX cotransfectants is shown (Gelatin). Merged staining (MT1-MMP/Gelatin) for the MT1-MMP/pcDNA3.0 cotransfectants is also shown. Transfected cells were visualized by wide-field fluorescence microscopy and acquired images were deconvoluted as described in Materials and Methods. Bars, 20 µm. (B) Steady-state distribution profile of MT1-MMP in A375 melanoma cells: A375 cells transfected with MT1-MMP and pcDNA3.0 or MT1-MMP and 1-PDX were biotinylated and incubated with TNE containing 1% Triton X-100 on ice. Soluble (S) and insoluble (P) fractions were separated by ultracentrifugation. Intracellular fractions (Int) were separated from plasma membrane fractions (PM) by capturing the biotinylated proteins with streptavidin-conjugated agarose beads. Each fraction was subjected to SDS-PAGE and transferred on nitrocellulose membrane. The MT1-MMP distribution profile was analyzed with immunopurified polyclonal anti-MT1-MMP antibody MMR2. The white arrowheads mark the positions of the pro- and mature (63 and 60 kDa) forms in the MT1-MMP/pcDNA cotransfectants, the black arrowheads indicate the 65 and 63 kDa immature forms in the MT1-MMP/1-PDX cotransfectants. Staining for caveolin-1 in the same experiment is reported as a bona-fide marker for the DRM preparation.