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Additional Supporting Information may be found in the online version of this article:

Figure S1: Localization of endogenous STAM proteins. A) HeLa cells were co-stained for endogenous STAM2 (green, left panels) and either the VTC marker ERGIC-53 (red, top) or the lysosomal marker LAMP1 (red, bottom). STAM shows a distribution pattern similar to that of ERGIC-53 but not LAMP1. An arrowhead identifies the region enlarged in the inset. B) Human embryonic kidney 293 cells (upper panels) and human astrocytes (bottom panels) were co-stained for endogenous STAM2 (green) and GM130 (red). The enlarged images (insets) reveal some colocalization, but staining patterns are not identical and appear juxtaposed. Bar, 10 μm.

Figure S2: Depletion of STAMs alters distribution patterns of Golgi markers. HeLa cells were transfected with control (upper panels) or STAM2 (lower panels) siRNA and co-stained with Golgi markers CIMPR and GGA3 or else the cytoskeletal markers actin (as assessed using phalloidin) or tubulin. As for GM130 (Figure 5C), the distribution pattern of each of these markers is much more condensed in STAM2 siRNA-transfected cells than in control cells. Actin and tubulin localizations are unchanged in STAM2 siRNA cells compared with control siRNA cells. Bar, 10 μm.

Figure S3: Suppression of STAM2 siRNA-induced, condensed Golgi phenotype by STAM2 overexpression. A) HeLa cells were transfected with control or STAM2-specific siRNAs, or else transfected with STAM2 siRNA and then retransfected with a modified, ‘knockdown resistant’ form of Myc-STAM2 (STAM2′) and then immunoblotted for STAM2 and Myc-epitope. Equal protein loading was monitored by immunoblotting for PLCγ. B) HeLa cells transfected with either control or STAM2 siRNA were then transfected with Myc-STAM2′. As observed for Myc-STAM2, overexpression of Myc-STAM2′ causes fragmentation of the Golgi apparatus in control cells (left panels). Lower levels of STAM2′ expression suppress the STAM2 siRNA-induced condensed Golgi phenotype in cells (low, middle panels); higher expression levels not only suppress the condensed Golgi phenotype but also cause Golgi fragmentation (high, right panels). C) The percentage of cells with condensed Golgi was quantified in control siRNA, STAM2 siRNA and STAM2 siRNA plus Myc-STAM2′-transfected cells; the proportion of cells with condensed Golgi in the siRNA recovery group (siRNA + STAM2′) was similar to that observed in control siRNA cells (= 3; 100 cells per experiment; ±SD).

Figure S4: EGF stimulation has no effect on Golgi morphology or ERES distribution in HeLa cells. A) HeLa cells were serum starved for 16 h, then stimulated with 100 ng/ml EGF and harvested at the indicated time-points. Cell extracts were immunoprecipitated with anti-phosphotyrosine antibodies and then immunoblotted with either anti-phosphotyrosine or anti-STAM1 antibodies. Peak STAM1 tyrosine phosphorylation occurred at about 10 min. B) HeLa cells were serum starved for 16 h and then stimulated with 100 ng/ml EGF for 0 or 10 min prior to fixation. Cells were immunostained for endogenous GM130, Sec16L and Sec31A. There were no significant differences in cellular distributions of these proteins.

Figure S5: Effects of Sec16L depletion on STAM2 distribution. A) Lysates from HeLa cells transfected for 72 h with any of three different Sec16L siRNAs or else control siRNA were immunoblotted for Sec16L. Equal protein loading was monitored by immunoblotting for PLCγ. B and C) HeLa cells were stained with antibodies against Sec16L (B) or else STAM2 (C) and GM130 (red, bottom panel) 72 h after transfection with control or Sec16L-specific siRNAs. The STAM2 distribution pattern in Sec16L-depleted cells is more punctate, and these puncta do not colocalize with the GM130 puncta (inset).

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FilenameFormatSizeDescription
TRA_856_sm_FigureS1.jpg281KSupporting info item
TRA_856_sm_FigureS2.jpg390KSupporting info item
TRA_856_sm_FigureS3.jpg226KSupporting info item
TRA_856_sm_FigureS4.jpg210KSupporting info item
TRA_856_sm_FigureS5.jpg289KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.