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Fig. 6. Effect of TMAO on GFP-PDS aggregates and on ER morphology. (A) Western blot analysis of the effect of TMAO on GFP-PDS and GFP-L236P turnover. Cells expressing GFP-PDS were incubated with CHX or TMAO for 1 hour. Cell lysates (15 µg) were probed with anti-PDS antibodies and anti-actin mAbs as a loading control. (B) Protease protection assay of TMAO- and CHX-mediated GFP-PDS aggregate dissociation. Cells expressing GFP-PDS were incubated with CHX or TMAO for 1 hour. Cell lysates were incubated with protease for 5 and 15 minutes and probed with anti-GFP mAb by western blotting. (C) Analysis of TMAO-mediated ER recovery using live-cell microscopy. Confocal images of cells expressing GFP-PDS were captured at 45-second intervals for 3 hours after the addition of 150 mM TMAO. Bar, 10 µm. See also Movie 3 in supplementary material.
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