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Fig. 5. Effects of drugs on the localization of TC48. (A) Cycloheximide-chase causes depletion of the Golgi pool of TC48. Cos-1 cells transfected with GFP-TC48 were allowed to express the exogenous cDNA for 12 hours at 37°C after which incubation was continued in the presence of cycloheximide (10 µg/ml) for 6 hours (Cyc+) and in the absence of cycloheximide (UT). Cells were fixed and stained with anti-giantin mouse monoclonal antibody (red) to stain the Golgi complex and observed by confocal microscopy (upper panels). Yellow colour in merged images indicates colocalization of TC48 and Golgi-localized giantin, and is evident in untreated cells (UT) but not in cells chased for 6 hours with cycloheximide (Cyc+). Notice, after 6 hours of cycloheximide-chase the Golgi pool of TC48 appears depleted. GFP-TC48 transfected and cycloheximide-treated or untreated cells were also stained with calnexin (ER marker) antibody (lower panels). (B) Nocodazole and BFA disturb the Golgi localization of TC48. Cos-1 cells expressing GFP-TC48 were treated, after 12 hours of transfection, with 5 µg/ml nocodazole at 37°C for 2 hours (Noc+) or with 5 µg/ml BFA for 30 minutes (BFA+). After fixation, cells were stained with giantin antibody and visualized by confocal microscopy. Bars, 10 µm.
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