Fig. 1. Active MT1-MMP is internalised in nonstimulated HT1080 cells. (A) The biotinylation experiment. Plasma membrane proteins were biotinylated on ice (black spots; biotinylation) and then internalised for 15 minutes at 37°C (uptake). Cells were cooled on ice and surface biotin was removed by treating the cells with MESNA for 25 minutes at 4°C as described in Materials and Methods (cleavage 1). Recycling of internalised biotinylated proteins was performed by placing the cells at 37°C for various times (chase), followed by a second MESNA treatment to cleave the biotin present on internalised proteins re-exposed at the cell surface (cleavage 2). (B) Active MT1-MMP is internalised in HT1080 cells. HT1080 cells were incubated on ice in the presence (lanes 2-5) or absence (lane 1) of biotin to label cell-surface proteins. Biotinylated HT1080 cells were kept for 15 minutes on ice (lanes 2 and 3) or warmed to 37°C (lanes 4 and 5) to allow internalisation and then treated with MESNA to remove cell-surface biotin (lanes 3 and 5). Biotinylated proteins were immunoprecipitated (IP) using an anti-biotin antibody, separated on an SDS-PAGE and analysed by immunoblotting using anti-MT1-MMP affinity-purified IgGs (IP). Unbound materials (non-IP) were also analysed. (C) bbHT1080 cells treated with (lanes 4, 5 and 6) or without (lanes 1, 2 and 3) PMA for 16 hours at 37°C were incubated with (lanes 2, 3, 5 and 6) or without biotin (lanes 1 and 4) on ice to label cell-surface proteins. Cells were warmed to 37°C to allow internalisation and treated with (lanes 3 and 6) or without (lanes 2 and 5) MESNA. Biotinylated proteins were immunoprecipitated, separated on an SDS-PAGE and analysed by immunoblotting.