Fig. 4. XBub3 antibodies override spindle checkpoint activation and maintenance in egg extracts. (A) Checkpoint activation. Aliquots of CSF-arrested Xenopus egg extract were pre-incubated with the following additions and then released from metaphase arrest with calcium chloride. Buffer control, nocodazole plus preimmune antibody, nocodazole plus rabbit XBub3 antibody, nocodazole plus XMad2 antibody. Chromosomes were visualised by the fluorescence of the DNA-binding dye Hoechst 33258 60 minutes after calcium addition. (B) Checkpoint activation. Samples were removed from the above egg extracts at the timepoints indicated for analysis of histone H1 kinase activity. The zero time point is histone H1 kinase activity prior to calcium addition. (C) Checkpoint maintenance. The spindle checkpoint was activated in aliquots of egg extract and the egg extracts further incubated at room temperature in the presence of a buffer control, nocodazole plus pre-immune antibody, nocodazole plus rabbit XBub3 antibody or nocodazole plus XMad2 antibody. Calcium was added to release the extracts from metaphase arrest and samples removed for analysis of H1 kinase activity at the timepoints indicated.