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Fig. 7. BFA does not impede ligand-mediated localization of S9(S+T)-CAD-GFP to the apicoplast. (A) Effect of BFA on apicoplast targeting. T. gondii stably transfected with S9(S+T)-CAD-GFP were grown for 3 days in the absence of ligand. Samples were incubated in one of several conditions prior to fixation as indicated: BFA, no ligand for 1 hour; BFA for 5 minutes followed by ligand for 20 minutes or 240 minutes; ligand alone for 20 minutes or 240 minutes. Samples were prepared for IFA, anti-GFP and quantum red streptavidin are shown, and a merge of anti-GFP, quantum red streptavidin and DAPI. (B) Effect of BFA on secretion. T. gondii stably transfected with S9(S)-CAD-GFP were grown overnight in the absence of ligand. GFP fluorescence in live cells was viewed at time 0 and then in the same cells 30 minutes after the addition of ligand or ligand plus BFA. As with apicoplast proteins, BFA was added 5 minutes before ligand. Arrow indicates parasitophorous vacuole. Lines were drawn on the same images to facilitate the localization of parasites and the parasitophorous vacuole membrane (lower panels). Bar, 5 µm.
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