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Fig. 5. Effect of inhibition of protein synthesis on folding and export of GFP-PDS. (A) Detection of CHX- and puromycin-mediated surface expression of GFP-PDS by biotinylation. Cells expressing GFP-PDS were incubated for 1 hour in culture medium containing 450 mM sucrose and 25 µg/ml CHX or 160 µg/ml puromycin. Cells were biotinylated using membrane-impermeable sulfo-NHS-biotin. Surface-labeled proteins were affinity-purified using avidin-agarose and probed by western blotting using anti-GFP and anti-actin mAbs as a loading control. (B) Confocal image analysis of CHX- or puromycin-mediated accumulation of YFP-PDS in the Golgi apparatus using a 20°C temperature block. Cells co-expressing YFP-PDS and the Golgi marker GalT-CFP were fixed after being incubated for 4 hours at 20°C in the presence of CHX or puromycin. Enlarged inserts show the perinuclear Golgi region. CFP- and YFP-tagged proteins were detected with Ar 458 nm and 514 nm laser lines, respectively. Bars, 10 µm.
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