Fig. 4. Active MT1-MMP is recycled to the cell surface in HT1080 cells. HT1080 cells were incubated on ice in the presence (lanes 2-9) or absence (lane 1) of biotin to label cell-surface proteins. Biotinylated cells were warmed to 37°C for 15 minutes to allow internalisation then cooled to 0°C and treated with (lane 3) or without (lane 2) MESNA. Cells were warmed to 37°C for 15 (lanes 4 and 5), 30 (lanes 6 and 7) and 45 minutes (lanes 8 and 9) to allow recycling and then treated with (lanes 5, 7 and 9) or without (lanes 4, 6 and 8) MESNA. Biotinylated proteins were immunoprecipitated, separated on an SDS-PAGE and analysed by immunoblotting using anti-MT1-MMP affinity-purified IgGs.