JCS02294 Supplementary Material

Files in this Data Supplement:

• Movie 1 -

  Movie 1. The effect of CHX on GFP-PDS-expressing cells. A movie of live cells incubated with 25 mg/ml CHX. Images were taken at 45-second intervals for 5.3 hours. Cells in a Labtek chamber were imaged on a heated stage using an inverted confocal laser-scanning microscope. A 253 0.8 NA objective was used with wide-open pinhole. The movie was captured using the “Advanced time series” macro set with embedded autofocus. Images were exported as TIFF, stacked, inverted, gamma-corrected and saved in Quicktime format using NIH Image 1.63 software (W. Rasband, NIH, Bethesda, MD).

• Movie 2 -

  Movie 2. The effect of CHX and ALLN on GFP-PDS-expressing cells. A movie of live cells incubated with 25 mg/ml CHX and 20 mg/ml ALLN. Images were taken at 45-second intervals. Other details are as in Movie 1.

• Movie 3 -

  Movie 3. Effect of TMAO on ER morphology. A movie of live cells incubated with 150 mM TMAO for several hours. Images were taken at 45-second intervals. Other details are as in Movie 1.

• Supplemental Figure 1 -

  Fig. S1. Intracellular distribution of PDS and GFP-PDS. (A) Collapsed ER in COS7 cells expressing untagged PDS shown by confocal image analysis. (B) GFP-PDS expression in HeLa cells shown by confocal image analysis. (C) Low-magnification confocal image of GFP-PDS expression in COS7 cells. (D) Colocalization of GFP-PDS with ER and PM markers. Confocal images of live cells expressing CFP-PDS with the ER marker sec61b-YFP (upper panel) or GPI-anchored YFP (lower panel). CFP- and YFP-tagged proteins were detected with Ar 458 nm and 514 nm laser lines, respectively. Bar, 5 mm.

• Supplemental Figure 2 -

  Fig. S2. Intracellular distribution of GFP-PDS at low expression levels. (A) Analysis of lysates from COS7 cells 16 hours after transfection with decreasing amounts of GFP-PDS plasmid DNA shown by western blot. (B)  Confocal images of live cells 16 hours after transfection with 0.14 and 0.004 mg/cm² plasmid DNA.

• Supplemental Figure 3 -

  Fig.S3. Expression of GFP-PDS in NRK cells. (A) Confocal image of stable NRK cells expressing GFP-PDS. (B) Different levels of GFP-PDS in transient and stable expression in COS7 and NRK cells respectively shown by dot-blot analysis.  Membrane was probed with anti-GFP mAb.

• Supplemental Figure 4 -

  Fig. S4. Induction of ER stress with tunicamycin. (A) Western blot analysis of the effect of tunicamycin (20 mg/ml) on COS7 cells expressing GFP-PDS or GFP-L236P. (B) Confocal image analysis of the effect of tunicamycin (20 mg/ml) on COS7 cells expressing GFP-PDS or GFP-L236P. Arrows show aggregated GFP-L236P. Bar, 20 mm.

• Supplemental Figure 5 -

  Fig. S5. Detection of CHX- and puromycin-mediated surface expression of GFP-PDS by biotinylation. Cells expressing GFP-PDS were incubated for 1 hour in the presence of 25 mg/ml CHX or 160 mg/ml puromycin without 450 mM sucrose. Cells were biotinylated using membrane-impermeable sulfo-NHS-biotin. Surface-labeled proteins were affinity-purified using avidin-agarose and probed by western blot using anti-GFP mAb.