Shugoshin Prevents Dissociation of Cohesin from Centromeres During Mitosis in Vertebrate Cells (original) (raw)
Figure 1
Centromeric Enrichment of Sgo1 in Mitosis
(A) Schematic overview of cell synchronisation and transfection procedure.
(B) Characterization of Sgo1 antibodies. Affinity-purified Sgo1 antibodies raised against two different regions of Sgo1 recognize the same 72-kDa protein by Western blotting. Synchronised HeLa cells were transfected with water (Mock) or Sgo1 siRNA. Cells were harvested 7 and 11 h after release from thymidine block, and chromatin fractions were resolved by SDS-PAGE and probed with antibodies 94 and 95. A major band at 72 kDa was detected by both antibodies, which disappeared or was greatly reduced upon Sgo1 knockdown. The blot was reprobed with HP-1β antibody as a loading control.
(C) Localisation of Sgo1. Cells were costained for Sgo1 with antibody 94 (shown in green), and with CREST antiserum (shown in red). DNA was counterstained with DAPI. Five different stages of mitosis are shown: (a) prophase, (b) prometaphase, (c) metaphase, (d) anaphase, and (e) telophase. Bar = 10 μm. (f) A single pair of CREST labelled kinetochores are enlarged, showing two adjacent Sgo1 foci as commonly observed for late stage metaphases. Bar = 1 μm.
(D) In situ immunofluorescence of (a) mock-treated or (b) Sgo1 siRNA-transfected HeLa cells, demonstrating the disappearance of the Sgo1 signal following siRNA treatment. The latter shows the typical in situ appearance of an Sgo1-depletion arrested cell (see Figures 3 and 6). Sgo1 was stained with antibody 94 (shown in green), and CREST is shown in red. DNA was counterstained with DAPI.