Supplemental Figures

This article contains the following supporting material:

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  Supplementary Figure 1: The localization of sialyltransferase (ST) was not altered by permeabilization and antibody treatment. HeLa cells grown on cover slips were transfected with ST-EGFP followed by permeabilization with Streptolysin O at 18°C and transport at 37°C in the presence of GS15 polyclonal antibody (A-C), GS28 monoclonal antibody (D-F) or control IgG (G-I), as described in the procedures of in vitro transport assay. The cells were then rinsed briefly, fixed and processed for immunofluorescence microscopy to reveal transfected ST-EGFP (A, D and G), and the presence of the added antibodies in each treatment, GS15 (B), GS28 (E), and control IgG (H). Bars, 10 μm.
  
  Supplementary Figure 2: The localization of tyrosylprotein sulfatransferase (TPST) was not altered by permeabilization and antibody treatment. (A-C) HeLa cells grown on cover slips were fixed directly and processed for immunofluorescence microscopy to reveal endogenous TPST (A) and GS28 (B). (D-I) HeLa cells grown on cover slips were subjected to permeabilization with Streptolysin O at 18°C and transport at 37°C in the presence of GS28 monoclonal antibody (D-F) or control IgG (G-I), as described in the procedures of in vitro transport assay. The cells were then rinsed briefly, fixed and processed for immunofluorescence microscopy to reveal endogenous TPST (D and G) and the presence of the added GS28 antibody (E) or control IgG (H) in each treatment. Bars, 10 μm. In addition to the typical perinuclear Golgi labeling of GS28 in panel E, the excess amount (200 μg/ml) of GS28 Mab also led to non-specific labeling of other non-Golgi structures in the perforated cells.
  
  Supplementary Figure 3: Overexpression of SNX3 partially arrested retrograde trafficking of CTxB at endosomal structures. HeLa cells transfected with Myc-SNX3 were allowed to internalize CTxB-Alexa Fluor 555 for 30min followed by a chase of 2.5 hours. SNX3 overexpressing cells (A-D) and control non-transfected cells (E-H) were subjected to triple labeling for expressed SNX3 (revealed by monoclonal anti-Myc and FITC-conjugated goat anti-mouse IgG), CTxB-Alexa Fluor 555, and the Golgi apparatus marked by Giantin (revealed by rabbit anti-Giantin and Alexa Fluor 647-conjugated goat anti-rabbit IgG). We observed that in SNX3 over-expressing HeLa cells, a small fraction of CTxB-AF555 could still be transported to the Golgi apparatus, although the majority of CTxB-AF555 is accumulated in the peripheral endosome-like structures. Bars, 10 μm.
  
  

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