Supplemental Figures

This article contains the following supporting material:

• Supplemental Figures 1-3.pdf - Supplemental Figure S1: Control siRNA experiments, demonstrating that the effects of Seh1 or Nup133 siRNA duplexes are not altered upon co-transfection with siRNAs targeting Giantin.
  (A) Widefield microscopy images of immunofluorescence of HeLa cells treated for 3 days with no siRNA (a, same as Fig. 4Ba) or with siRNA duplexes specific for Seh1 (b) or Nup133 (c) and labeled with anti-Nup107 antibody, mAb414, anti-Giantin scFv, and DAPI.
  (B) Widefield microscopy images of immunofluorescence of HeLa cells treated for 3 days with no siRNA (a), with siRNA duplexes specific for Seh1 (b) or Nup133 (c), or cotransfected with siRNA duplexes specific for Nup133 and for Giantin. Cells were labeled with anti-Tpr, anti-SC-35, and anti-Giantin antibodies, and stained with DAPI. Bars, 10 μm. Note that the phenotypes induced by Seh1 or Nup133 siRNA duplexes alone are indistinguishable from those obtained upon co-transfection with siRNA duplexes specific for Giantin (compare Fig S1A b with Fig 4B d, Fig S1A c with Fig 4B c, Fig S1B b with Fig 4D c, and Fig S1B c with Fig. S1B d). Conversely, Giantin labeling is not altered upon treatment with siRNA duplexes specific for Seh1 or Nup133, thereby validating the use of siRNAs targeting Giantin to follow the co-transfected Nup133 or Seh1 siRNAs.
  
  Supplemental Figure S2: GFP-Nup43 is localized at kinetochores from prophase to anaphase.
  (A) Deconvolved images of fixed metaphase HeLa cells expressing GFP-Nup43 (green) and labeled with anti-CENP-F antibody (red) and the CREST serum (blue). Inserts show enlargements of the marked area.
  (B) Fixed HeLa cells expressing GFP-Nup43 were labeled with the CREST serum and DAPI. Deconvolved images of interphase (a), prophase (b), early anaphase (c) and late anaphase (d) are presented. The insets show enlargements of the marked area. Arrows point to the position of centromeres or kinetochores.
  
  Supplemental Figure S3: Unlike Sec13, Sec31 does not localize to the nuclear envelope or kinetochores. (A) Deconvolved images of GFP₃-Sec13 expressing cells labeled with anti-Sec31 antibody. Arrowheads point to the specific labeling of GFP₃-Sec13 at the kinetochore (top) and nuclear envelope (bottom). Arrows indicate structures in which the GFP₃-Sec13 and the anti-Sec31 labeling overlap in interphase and mitotic cells.
  (B) Deconvolved images of HeLa cells double-labeled with affinity-purified anti-Sec31 antibody and the CREST serum. DNA was stained with DAPI. Arrowheads point to the position of kinetochores, labeled with the CREST serum, but not with the Sec31 antibody, and arrows to fluorescent Sec31 foci which are not labeled by the CREST serum.
  

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