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Fig. 1. Intracellular distribution of GFP-PDS and effect on ER function in COS7 cells. (A) Confocal images of fixed cells expressing untagged PDS, and living cells expressing GFP-PDS and GFP-CFTR, 16 hours after transfection. Cells expressing untagged PDS were fixed, permeabilized and probed with anti-PDS antibody and secondary Cy3 anti-rabbit antibody as described in Materials and Methods. GFP and Cy3 were detected with Ar 488 nm and HeNe 543 nm laser lines, respectively. Arrows indicate the plasma membrane (PM), endoplasmic reticulum (ER) and perinuclear aggregate (AGG). Bars, 10 µm. (B) Perinuclear GFP-PDS forms aggregates as demonstrated by photobleaching. A rectangle in the middle of a perinuclear aggregate was photobleached in a cell expressing GFP-PDS using a high-power 488 nm laser. Bars, 10 µm. (C) Detection of surface GFP-PDS and GFP-L236P by biotinylation. 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 mAb. Results of quantitative densitometry are presented, on the right, as percentage of the sum of total lysate and low speed (14K) pellet. (D) Western blot analysis of the effect of GFP-PDS, GFP-L236P, GFP-CFTR and GFP-
508 expression on proteasomal degradation. Cells were transfected with equal amounts of plasmids as described in Materials and Methods and harvested after 16 hours. Western blot analysis showing the accumulation of ubiquitilated proteins as a result of overexpression of GFP-tagged proteins probed with anti-GFP (top panel) or anti-ubiquitin (bottom panel) antibodies. (E) Effect of co-expressed GFP-PDS on GFP-VSVG surface-biotinylation labeling. Cells expressing GFP-VSVG in the presence or absence of GFP-PDS were preincubated overnight at 40°C prior to 2 hours incubation at 32°C. 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 mAb. Biotinylated GFP-VSVG bands are indicated with an asterisk.
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