JCS036103 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Depletion of syntaxin 18 induces Golgi disassembly. HeLa cells were treated as described in the legend to Fig. 1A and stained with an antibody against GM130, Man II, β-COP or KDEL-R. Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Time course of morphological changes in the ER, Golgi, ERGIC and ER exit sites in cells transfected with Syn18 (390). At the indicated times after transfection, HeLa cells were double-stained with antibodies against Bap31 and ERGIC-53, Sec61β and PDI, KDEL-R and GM130, or Sec31A and β-COP. A ×100 objective lens was used to obtain the images. Scale bar: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Ultrastructure of a syntaxin 18-depleted cell. HeLa cells were treated as described in the legend to Fig. 2. Arrows, arrowheads and asterisk indicate vesiculated membrane structures, ER aggregates and dilate ER, respectively. Scale bar: 500 nm.

• Supplemental Figure S4 -

  Fig. S4. Immunoelectron microscopy of ER and Golgi proteins in mock-treated cells. HeLa cells were mock-transfected, incubated for 72 hours and processed for immunoelectron microscopic analysis. Localization of a cis-Golgi marker, p115 (A), a trans-Golgi marker, Vti1a (B), Bap31 (C), CLIMP-63 (D) and Hsp47 (E) N, nucleus and G, Golgi complex. Arrowheads indicate typical ER. Bars, 500 nm.

• Supplemental Figure S5 -

  Fig. S5. Transport of VSVG-GFP from the ER is slightly slowed by syntaxin 18 depletion. The rate of VSVG-GFP transport from the ER was determined by immunofluorescence microscopy (A) and by the acquisition of endoglycosidase H resistance (B). The boxed areas are shown enlarged. Scale bar: 10 µm. The morphological and biochemical transport assays were performed as described by Iinuma et al. (2007).

• Supplemental Figure S6 -

  Fig. S6. Expression of ERGIC-53 and localization of GFP-ERGIC-53 in syntaxin 18-depleted cells. (A) HeLa cells were mock-transfected or transfected with Syn18 (390) and incubated for 72 hours. The cell lysates (15 µg) were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (B) At 48 hours after transfection of siRNA, the plasmid encoding GFP-ERGIC-53 (Ben-Tekaya et al., 2005) was transfected, and the cells were further incubated for 24 hours, fixed and observed by fluorescence microscopy. A ×100 objective lens was used to obtain images. Scale bar: 10 µm.

• Supplemental Figure S7 -

  Fig. S7. ERGIC-53 is not returned to the ER in syntaxin 18-depleted cells. HeLa cells were transfected with Syn18 (390), incubated for 72 hours and processed for immunoelectron microscopic analysis. N, nucleus. Arrowheads and arrows indicate typical ER and dilated ER, respectively. The structures labeled with asterisks are probably aggregated ER structures. Perhaps aggregated ER membranes were not completely fixed with paraformaldehyde. No immunoreactivity of ERGIC-53 was detected in ER or dilated ER structures. Scale bar: 2 µm.

• Supplemental Figure S8 -

  Fig. S8. Redistribution of ERGIC-53 by syntaxin 18 depletion is not suppressed by knockdown of Rab6. HeLa cells were mock-transfected or transfected with lamin A/C siRNA, Rab6 siRNA, Syn18 (390) or both Rab6 siRNA and Syn18 (390), and incubated for 72 hours. (A) The cells were solubilized in phosphate-buffered saline with 0.5% SDS, and the lysates (10 µg each) were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The target proteins were fully knocked down. (B-D) The cells were fixed and stained with an antibody against p115 (B), ERGIC-53 (C) or Man II (D). The distributions of the marker proteins examined were indistinguishable between mock-treated cells and lamin A/C siRNA-treated cells (data not shown). Scale bars: 10 µm.

• Supplemental Figure S9 -

  Fig. S9. BFA induces redistribution of the Golgi GPP130 to the ER in syntaxin 18-depleted cells. HeLa cells were treated as described in the legend to Fig. 9. A ×100 objective lens was used to obtain images. Scale bar: 10 µm.

• Supplemental Figure S10 -

  Fig. S10. BFA effect is reversible. HeLa cells were treated as described in the legend to Fig. 9. BFA was washed out, and the cells were incubated at the indicated times and processed for immunofluorescence microscopy. ERGIC-53-negative large puncta observed at 3 hours after BFA washout in Syn18 (390)-transfected cells probably correspond to ER patches. A ×100 objective lens was used to obtain images. Scale bar: 10 µm.