Fig. 5. GLT-1 is targeted to degradative acidic compartments in G93A-expressing MDCK cell lines. Forty-eight hours after GLT-1 transfection, the cells were fixed and stained for EEA1 (green), a marker of early endosomal compartments, and GLT-1 (red). To verify whether GLT-1 accumulates in the sub apical slow recycling compartment labelled by the mutant 5BGT epithelial GABA transporter (Perego et al., 1999), the G93A-expressing MDCK cells were co-transfected with GLT-1 and myc-tagged 5BGT, and fixed and double-stained for GLT-1 (red) and myc (green) after 48 hours. Confocal analysis of the double indirect immunofluorescence staining showed no colocalisation of GLT-1 with EEA1 or 5BGT, whereas colocalisation of GLT-1 (yellow) with acidic organelles was revealed by labelling with the acidotropic FITC-lysotracker (0.5 mM) for 1 hour at 37°C before fixation and staining for GLT-1. Bar in upper panels, 5 µm; middle panels, 2 µm; lower panels, 3 µm.