Fig. 3. GFP-ST localises both to the cell surface and to the Golgi complex. (A) Confocal analysis of CV-1 cells expressing GFP-ST and stained either for surface glycoproteins with Con A (a-c) or immunostained for giantin (d-f) (see Materials and Methods). Single confocal sections viewed under the fluorescein filter for GFP (a, d) and under the Texas Red filter for surface glycoproteins (b) or for the Golgi complex (e) are shown. The resulting merged images are shown in (c) and (f), respectively. The yellow colour indicates colocalisation. Bar, 10 µm. Inset in panels (d-f) is magnified 2.5 times. (B) Golgi localisation of GFP-ST is not affected by the level of expression of the construct and decreases after cycloheximide treatment. Transfected cells were either fixed or first incubated with cycloheximide (50 µg/ml) for 4 hours before fixation. After immunostaining with anti-giantin antibodies, Z series were acquired (see Materials and Methods). Average fluorescence and the percentage of total fluorescence colocalizing with giantin were determined for 25 cells of each group. The mean percentage of GFP in the Golgi is given for groups of cells classified according to their summed average fluorescence intensity over the stack. The numbers inside the columns indicate the number of cells in each group. Gray columns, untreated cells; pink columns, cells treated with cycloheximide; bars indicate the standard deviation.

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