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	Figure 1. BubR1 and its kinase activity are essential for metaphase chromosome alignment in Xenopus egg extracts. (A, B, D, E, H, and I) Representative mitotic structures formed in the presence of IgY (A) and BubR1 antibody (B) or mock depleted (D), BubR1 depleted (E), and BubR1 depleted and supplemented with recombinant WT-BubR1 (H) or KD-BubR1 (I). (C and J) Quantification of structures formed from sperm nuclei in cycled egg extracts as indicated scored 80 min after exit from interphase. At least 50 mitotic structures were scored for each extract. Data are presented from one representative experiment. (F and G) Mitotic structures formed in egg extracts treated with a low dose of nocodazole (100 nM) for 10 min. (K) CSF egg extracts were immunodepleted of BubR1 using purified IgY or BubR1 antibodies, and BubR1 protein was assayed by immunoblotting. (L) Purification of recombinant Xenopus WT- and KD-BubR1. Initial E. coli lysates encoding GST–WT-BubR1 (lane 1) and GST–KD-BubR1 (lane 3) and GST–WT-BubR1 (lane 2) and GST–KD-BubR1 (lane 4) after affinity purification over immobilized glutathione. Bars, 10 μm.
	Figure 2. BubR1 forms a complex with APC/EB1 in Xenopus egg extracts. (A and B) Immunoprecipitates from CSF-arrested egg extracts containing sperm nuclei with affinity-purified chicken anti-BubR1 IgY (A) or rabbit anti-APC IgG (B) antibodies probed with BubR1, CENP-E, APC, or EB1 antibodies. FL, extracts after immunoprecipitation; IP, immunoprecipitates. A threefold higher proportion of the bead-bound fraction relative to the depleted extracts was analyzed. (C) Cofractionation of BubR1 with APC and EB1 after sucrose density gradient centrifugation. The high speed supernatant of metaphase egg extracts was separated over a 5–15% continuous sucrose gradient. Fractions were collected and analyzed by immunoblotting with the indicated antibodies. (D) Indirect immunofluorescence image of APC (green), BubR1 (red), and DNA (blue) in cycled egg extracts. APC and BubR1 are shown magnified in the insets. (E) Immunoprecipitates from CSF-arrested egg extracts containing or not containing sperm nuclei and nocodazole (as indicated) with affinity-purified rabbit anti-APC IgG probed with APC (top), BubR1 (middle), or EB1 (bottom) antibodies. Immunoreactivity was quantified relative to the value in lane 1. (F) Characterization of APC and EB1 antibodies. CSF-arrested egg extracts were immunoblotted with preimmune (lanes 1 and 3) or anti-APC or -EB1 antibodies (lanes 2 and 4).
	Figure 3. APC and EB1 depletion results in chromosome alignment defects. (A–F) Metaphase spindles assembled in mock- (A), APC- (B), EB1- (D), or APC/EB1 (F) -depleted egg extracts and APC- or EB1-depleted egg extracts supplemented with purified recombinant APC (C) or EB1 (E). (G) Quantification of structures formed from sperm nuclei in cycled egg extracts as indicated scored 80 min after exit from interphase. At least 50 mitotic structures were scored for each extract. Data are presented from one representative experiment. (H and I) Immunoblot of immunodepleted CSF egg extracts. CSF-arrested egg extracts were immunodepleted of APC or EB1 using APC and EB1 antibodies. 1 μl of control extracts with serial dilution (as indicated) or 1 μl of depleted extracts was analyzed for APC (H) or EB1 (I) levels. (J and K) Purification of recombinant Xenopus APC (J) and EB1 (K). (J) An initial whole cell extract from insect cells infects with a baculovirus encoding His-APC (lane 1) and purified APC (lane 2) after purification over immobilized Ni-NTA agarose. (K) An initial E. coli lysate encoding GST-EB1 (lane 1) and GST-EB1 after affinity purification over immobilized glutathione (lane 2). (L–N) Mitotic structures formed in egg extracts treated with a low dose of nocodazole (100 nM) for 10 min. Bars, 10 μm.
	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).
	Figure 5. BubR1–APC/EB1 complex formation is important for metaphase chromosome alignment. (A–H) Metaphase spindle structures assembled in mock- (A and E), BubR1/APC- (B), BubR1–APC/EB1- (C), APC/Aurora B- (D), BubR1/Aurora B- (E), and APC/MCAK (F) -depleted egg extracts or egg extracts supplemented with purified recombinant N-APC (G) or EB1C (H). (I) Quantification of structures formed from sperm nuclei in cycled egg extracts as indicated scored 80 min after exit from interphase. At least 50 mitotic structures were scored for each extract. Data are presented from one representative experiment. (J and K) Purification of recombinant Xenopus N-APC (APC1–1,450; J) and GST-EB1C (EB1165–268; K). Initial E. coli lysates encoding N-APC or EB1C (lane 1) and GST–N-APC or GST-EB1C after affinity purification over immobilized glutathione (lane 2). (L) Immunoprecipitates with an APC antibody from CSF-arrested egg extracts containing sperm nuclei (lanes 3 and 4) or supplemented with N-APC (lanes 1 and 2) or EB1C (lanes 5 and 6) with affinity-purified rabbit anti-APC IgG and probed with APC, BubR1, EB1, or GST (for GST–N-APC and GST-EB1C) antibodies. FL, extracts after immunoprecipitation; IP, immunoprecipitates. A threefold higher proportion of the bead-bound fraction relative to the depleted extracts was analyzed. Bars, 10 μm.
	Figure 6. BubR1 phosphorylates APC and forms a ternary complex with APC and microtubules in vitro. (A–C) In vitro binding assay using purified proteins as indicated. Purified recombinant proteins were incubated with glutathione–Sepharose 4B (A and C) or Ni-NTA agarose (B) beads as indicated. Protein pulldown with the beads was assayed by SDS-PAGE and Coomassie blue staining. (D) APC phosphorylation by BubR1. In vitro kinase assay was performed with a combination of purified recombinant APC (lane 7), BubR1 (lane 8), and CENP-E (lane 9) as indicated. (E) BubR1, APC, and microtubules form a ternary complex. After centrifugation through a 40% sucrose cushion, BubR1–APC–taxol microtubule complex formation was assayed by immunoblotting. S, supernatant; P, pellet.
	Figure 7. BubR1 kinase activity is essential for the efficient recruitment of APC onto kinetochores in Xenopus egg extracts. (A) BubR1-depleted egg extracts containing sperm nuclei were cycled through interphase and arrested with CSF activity at the following metaphase. Recombinant WT-BubR1 (top) or KD-BubR1 (bottom) was added. Kinetochore recruitment of BubR1 (left; red), APC (right), and microtubules (left; green) was visualized by immunofluorescence with specific antibodies. Chromatin was visualized with DAPI (left; blue). (B) Quantification of the relative BubR1 and APC intensity at kinetochores in A. Error bars represent SEM. (C) Immunoprecipitates with an APC antibody from BubR1-depleted CSF egg extracts containing sperm nuclei and supplemented with purified WT-BubR1 (lanes 1 and 2) or KD-BubR1 (lanes 3 and 4) and probed with APC (top), BubR1 (middle), and EB1 (bottom) antibodies. FL, extracts after immunoprecipitation; IP, immunoprecipitates. A threefold higher proportion of the bead-bound fraction relative to the depleted extracts was analyzed. (D) Model: BubR1 is recruited onto unattached kinetochores. Microtubule-associated proteins APC/EB1 bind to the plus ends of microtubules. After the initial capture of microtubules by kinetochores, the interaction between BubR1 and APC/EB1 stabilizes kinetochore microtubule attachment, in which BubR1 might directly phosphorylate APC. Bar, 10 μm.

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