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Fig. 4. GFP-PDS is ubiquitilated. (A) Immunofluorescence co-localization of GFP-PDS with ubiquitin. Confocal images of cells expressing GFP-PDS were treated with ALLN for 6 hours. Cells were fixed, permeabilized and probed with anti-ubiquitin mAb and secondary anti-mouse Cy3-tagged mAb. GFP and Cy3 were detected with Ar 488 nm and HeNe 543 nm laser lines, respectively. Magnified views on the left show aggregates of ubiquitilated (CK SP) GFP-PDS. Arrowheads in the left panels point to a non-GFP-PDS-expressing cell. Arrows indicate the nucleus. Boxed regions in the center panels are magnified fivefold to show perinuclear aggregates. N, nucleus; AG, aggregate. Bar, 20 µm. (B) Western blot analysis showing ubiquitilation of GFP-PDS by immunoprecipitation. Cells expressing GFP-PDS were incubated for 1 hour with ALLN. Anti-GFP mAb-GFP-PDS complexes were immunoisolated (IP) from lysates using protein G agarose and probed with anti-ubiquitin mAb. (C) Live-cell microscopy of CHX-mediated recovery of GFP-PDS-expressing cells in the presence of ALLN. Representative images from a time-lapse series of cells expressing GFP-PDS, incubated in the presence of 25 µg/ml CHX and 20 µg/ml ALLN for 3.5 hours. Images were captured at 45-second intervals. Arrows indicate GFP-PDS in aggregates and in collapsed ER. Bar, 20 µm. See also Movie 2 in supplementary material.
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