Figure 2.

Bub1 is necessary for the establishment and maintenance of the spindle checkpoint in frog egg extract. (A) Anti-Bub1 antibodies abolish the spindle checkpoint. CSF-arrested extracts were incubated for 30 min on ice without addition of anti-Bub1 antibody (top), with anti-Bub1 antibodies (middle), or with anti-Bub1 antibodies preblocked with recombinant Bub1 protein (bottom). Sperm nuclei and nuclei were added for 20 min, followed by the addition of calcium chloride to overcome the metaphase arrest. Samples were taken immediately before (t = 0) the addition of calcium and every 15 min thereafter, and histone H1 kinase activity was determined. Autoradiograms of histone H1 kinase assay as well as photographs of chromosomes at times indicated at bottom are presented. (B) Anti-Bub1 antibodies abolish the establishment and maintenance of the spindle checkpoint. For the effect on checkpoint establishment, CSF-arrested extracts were preincubated with control IgG (top), anti-Bub1 (middle), or anti-Mad1 antibodies (bottom) as described in A. For the effect on checkpoint maintenance, checkpoint was first established in extracts with sperm nuclei and nocodazole, followed by the addition of antibodies. Samples were taken for histone H1 kinase assay as in A. The decline in H1 kinase activity was accompanied by decondensation of chromosomes (data not shown). Bar, 10 μm.