Figure 8.

Cohesin stays on the chromosomes in mitotic cells lacking Separase. A Separase ^Δ/flox stable cell line expressing Scc1-myc was generated. (A–C) Characterization of the cell line. (A) Silver stain of the IP products from cells expressing Scc1-myc and control cells using an anti-myc antibody. (B) Western blot of the same IP reaction as in A using anti-myc and anti-Smc3 antibodies. E, eluate; S, supernatant; ce, cell extract. (C) Immunostaining using anti-myc antibody (green) and CREST serum (red). Cells were treated with nocodazole for 30 min and spun on glass slides. Note that Scc1-myc staining was observed in the centromeric region between two CREST dots. (D) Cells were infected with the virus as in Fig. 5 A, and 24 h after splitting cells were processed for immunofluorescence microscopy. Mitotic cells were spun on glass slides and analyzed for cohesin with an antibody to the myc epitope and P-H3. (E) Cells positive for myc and P-H3 as well as negative for myc but positive for P-H3 were scored. n = 200 per cell type. (F) Cells were infected in the same way as in D and collected 48 h after splitting. Before harvesting, cells were treated with nocodazole for 30 min to disrupt the spindle, leading to better spreading of chromosomes. Bars, 10 μm.