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Fig. 3. Effect of inhibition of protein synthesis on solubility and intracellular distribution of GFP-PDS. (A) Live-cell analysis of CHX-mediated recovery of GFP-PDS-expressing cells. Representative images of a time-lapse sequence of cells expressing GFP-PDS incubated in the presence of 25 µg/ml CHX for 5.3 hours. Images were captured at 45-second intervals. Arrows indicate cells with recovering ER. Right: regions of interest of total cellular fluorescence are plotted against time for the three cells outlined in the 0 minute panel. Bars, 20 µm. See also Movie 1 in supplementary material. (B) Live-cell microscopy of puromycin-mediated redistribution of the ER-retained GFP-PDS. Confocal images of cells expressing GFP-PDS incubated for 6 hours with or without 160 µg/ml puromycin. Bars, 10 µm. (C) Western blot analysis of the effect of CHX on GFP-PDS solubilization and degradation. Cells expressing GFP-PDS were incubated with increasing concentrations of CHX for 6 hours. Cell lysates (15 µg protein, top panel) and the 14K pellet (total pellet loaded, bottom panel) were probed with anti-PDS antibodies. (D) CHX-mediated degradation of GFP-PDS and GFP-L236P by western blot. Cells expressing GFP-PDS or GFP-L236P were incubated for the designated times with 25 µg/ml CHX. Cell lysates (15 µg protein) were probed with anti-PDS antibodies.
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