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. 2007 Aug 27;178(5):773–784. doi: 10.1083/jcb.200702138

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Copyright © 2007, The Rockefeller University Press

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Figure 4. — APC/EB1 depletion arrests Xenopus egg extracts in mitosis. (A) CSF-arrested Xenopus egg extracts were mock depleted (a and b), BubR1 (c), APC (d and e), or EB1 (g and h) depleted, or APC or EB1 depleted and supplemented with recombinant APC (f) and EB1 (i). After incubation of sperm nuclei with or without nocodazole (as indicated), CSF activity was inactivated by the addition of calcium. Aliquots were taken from each extract at the indicated times and assayed by autoradiography for Cdk1 kinase activity (left) using added histone H1 as a substrate and (right) maintenance of chromatin condensation. (B) The depletion of APC and EB1 activates BubR1 kinase in CSF-arrested Xenopus egg extracts. After immunodepletion of endogenous BubR1, recombinant GST-BubR1 was added to a molar level comparable with the endogenous level of BubR1 in CSF-arrested egg extracts. Sperm nuclei and nocodazole were then added to mock-, APC-, or EB1-depleted egg extracts or supplemented with recombinant APC and EB1 as indicated. After 30 min, GST-BubR1 was immunoprecipitated using specific anti-GST antibodies and immunoblotted with anti-BubR1 antibody (bottom) or kinase activity assayed after the addition of histone H1 and γ-[³²P]ATP (top). (C) CSF-arrested egg extracts were mock depleted or BubR1, APC, and EB1 depleted. After the addition of sperm nuclei and nocodazole, extracts were observed by indirect immunofluorescence with anti-BubR1 (green) and Mad2 (red) antibodies, and chromatin was visualized with DAPI (blue).