Fig. 6. MT1-MMP internalisation is greatly reduced in cells with perturbed caveolae function. bbHT1080 cells treated in the absence (A,C) or in the presence of 30 µg/ml nystatin (B) or 4 mM methyl-ß-cyclodextrin (D) were subjected to an antibody uptake assay using anti-MT1-MMP affinity-purified IgGs. After 15 minutes, cells were fixed and TX-100 permeabilised, and IgG-bound MT1-MMP complexes were revealed using a FITC-conjugated secondary antibody. Arrowheads are examples of internalised MT1-MMP-positive structures. Bar, 20 µm. (E) bbHT1080 cells were not treated (lanes 2 and 3) or treated for 1 hour with 30 µg/ml nystatin (lanes 4 and 5) or for 16 hours with 50 µg/ml concanavalin A (lanes 6 and 7) before cell-surface biotinylation as described in Materials and Methods. After 15 minutes internalisation, cells were treated with (lanes 3, 5 and 7) or without (lanes 2, 4 and 6) MESNA to remove cell-surface biotin. Biotinylated proteins were immunoprecipitated, separated on a SDS-PAGE and analysed by immunoblotting using anti-MT1-MMP affinity-purified IgGs.