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	Figure 1. BubR1 is a phosphoprotein associated with Bub3. (A) Specificity of the anti-BubR1 antibody. Interphase (lanes 1 and 4), metaphase (lanes 2 and 5), or spindle checkpoint–active (lanes 3 and 6) extracts were immunoblotted with anti-BubR1 antibody (lanes 4–6) or the same antibody preincubated with recombinant BubR1 protein (lanes 1–3). The migration of molecular standards is indicated on the left. (B) Anti-BubR1 antibody recognizes BubR1, but not Bub1. Immunoprecipitation was performed from metaphase extracts with a control IgG (lanes 1 and 4), anti-Bub1 (lanes 2 and 5), or anti-BubR1 (lanes 3 and 6) antibody. The supernatants left after immunoprecipitation (lanes 1–3) or the immunoprecipitates (lanes 4–6) were probed with anti-BubR1 (top) or anti-Bub1 (bottom) antibody. The migration of molecular standards is indicated on the left. (C) BubR1 is a phosphoprotein. Interphase (I; lanes 1, 4, and 7), metaphase (M; lanes 2, 5, and 8), and spindle checkpoint–active (N; lanes 3, 6, and 9) extracts were treated with phosphatase buffer (lanes 1–3), LPP (lanes 4–6), or LPP and phosphatase inhibitors (lanes 7–9). The extracts were then immunoblotted with anti-BubR1 antibody. The migration of molecular standards is indicated on the left. (D) BubR1 associates with Bub3. Anti-BubR1 immunoprecipitates were prepared from interphase (lanes 1 and 5), metaphase (lanes 2 and 6), or spindle checkpoint–active (lanes 3 and 7) extracts, or from Bub1-depleted extract with the spindle checkpoint provoked (lanes 4 and 8). The extracts (lanes 1–4) or the immunoprecipitates (lanes 5–8) were immunoblotted with anti-BubR1 (top) or anti-Bub3 (bottom) antibody.
	Figure 2. Mutual dependency between BubR1 and Mad2 in binding to Cdc20. CSF-arrested extracts were depleted with control IgG (mockΔ; lanes 1–3 and 10–12), anti-BubR1 (BubR1Δ; lanes 4–6 and 13–15), or anti-Mad2 (Mad1/2Δ; lanes 7–9 and 16–18) antibody. The extracts were induced to enter interphase (lanes 1, 4, 7, 10, 13, and 16), were kept at metaphase (lanes 2, 5, 8, 11, 14, and 17), or were incubated with nuclei and nocodazole to activate the spindle checkpoint (lanes 3, 6, 9, 12, 15, and 18). Anti-Cdc20 immunoprecipitates were prepared from these extracts. The extracts (lanes 1–9) or immunoprecipitates (lanes 10–18) were immunoblotted for the proteins indicated on the left. The asterisks indicate a cross-reacting protein and IgG heavy chain on the left and right panels of Cdc20 blots, respectively. The asterisk on the right of Mad2 blot indicates IgG light chain that migrates right above Mad2. Immunodepletion with anti-Mad2 antibody removes both Mad1 and Mad2.
	Figure 3. BubR1 is required for kinetochore binding of Mad1, Mad2, Bub1, and CENP-E. Mitotic chromosomes were isolated from mock- or BubR1- depleted extracts treated with nocodazole and then incubated with rabbit antibodies against Mad1, Mad2, or Bub1, as indicated on top, followed by fluorescein-conjugated anti–rabbit antibody. BubR1 was then detected with biotinylated anti-BubR1 antibody and Texas red– conjugated streptavidin. For double staining of CENP-E and BubR1, rabbit antibody against BubR1 was used, followed by fluorescein-conjugated anti–rabbit antibody. CENP-E was then detected with biotinylated anti–CENP-E antibody and Texas red–conjugated streptavidin. Chromosomes were stained with Hoechst 33258. The merge pictures contain all three fluorochromes. All images of the same fluorochrome were taken for the same exposure time and processed in the same way. Bar, 10 μm.
	Figure 4. BubR1 is required for other spindle checkpoint proteins to associate with chromosomes. (A) Immunoblot analysis of various checkpoint proteins associated with chromosomes. Mitotic chromosomes were isolated from mock-depleted (lanes 1, 2, 5, and 6) or BubR1-depleted (lanes 3, 4, 7, and 8) extracts that were untreated (lanes 1, 3, 5, and 7) or treated with nocodazole (lanes 2, 4, 6, and 8). The extracts or chromosomal fractions were immunoblotted for proteins indicated on the left. The migration of molecular size standards is indicated on the right for BubR1 and Bub1 blots. (B) Phosphatase treatment removes mobility shift of chromosomal BubR1 and Bub1. Chromosomal pellets prepared from untreated extracts (lanes 1, 3, 5, and 7) or extracts treated with nocodazole (lanes 2, 4, 6, and 8) were treated with phosphatase buffer (lanes 1, 2, 5, and 6) or LPP (lanes 3, 4, 7, and 8). The samples were then immunoblotted for BubR1 (lanes 1–4) or Bub1 (lanes 5–8). The migration of molecular size standards is indicated on the right.
	Figure 5. BubR1 is required for spindle checkpoint independently of its kinase activity. (A) Autoradiographs of histone H1 kinase assay. Extracts were depleted with a control IgG (mock depleted), or with anti-BubR1 antibody, and then supplemented with mock, wild-type BubR1, BubR1KR, or BubR11–742 as indicated on the left. The level of BubR1 protein in extracts was similar to that shown in B (lanes 1–5). After incubation with nuclei and nocodazole, calcium was added to the extracts to inactivate CSF activity and to trigger mitotic exit. Samples were taken immediately before calcium addition (time = 0) or every 15 min thereafter as indicated on bottom. (B) Extracts were depleted with a control IgG (lanes 1 and 6) or anti-BubR1 antibody and then supplemented with mock (lanes 2 and 7), wild-type BubR1 (lanes 3 and 8), BubR1KR (lanes 4 and 9), or BubR11–742 (lanes 5 and 10). The spindle checkpoint was induced in these extracts by incubation with nuclei and nocodazole, followed by anti-Cdc20 immunoprecipitation. The extracts (lanes 1–5) or the immunoprecipitates (lanes 6–10) were immunoblotted for proteins indicated on the left. Asterisks indicate a cross-reacting protein and IgG heavy chain on the left and right panels, respectively.
	Figure 6. Localization of BubR1 to kinetochores is dependent on Bub1 and Mad1. (A) Bub1 is required for BubR1 to associate with kinetochores. Mitotic chromosomes were isolated from mock- or Bub1-depleted extracts in the presence of nocodazole and subjected to immunofluorescence staining for BubR1 and Bub1 as described for Fig. 3. The level of depletion is similar to that shown in Fig. 1 A. (B) Kinetochore association of BubR1 is reduced in the absence of Mad1. Extracts were mock depleted or depleted for both Mad1 and Mad2 (Mad1/2 depletion). The latter extracts were then supplemented with mock, Mad1, Mad2, or both Mad1 and Mad2 as indicated on the left. Mitotic chromosomes were assembled in these extracts lacking or containing nocodazole as indicated on the right. Chromosomes from each sample were isolated and loaded onto two coverslips. One set of the samples was stained for BubR1 and Mad1, the other for BubR1 and Mad2 as described for Fig. 3. For space conservation, BubR1 staining was shown for only one set of the samples. Bar, 10 μm. Immunoblot analysis of the extracts is similar to Fig. 7 (lanes 1–6).
	Figure 7. Hyperphosphorylation of chromosomal BubR1 requires Mad1. Mock-depleted extracts or extracts lacking Mad1 and/or Mad2 were prepared as described for Fig. 6 B (lanes 1–6). The extracts were incubated with nuclei (15,000 nuclei/μl extract) in the absence or presence of nocodazole as indicated on top. The samples were then subjected to chromosome isolation or immunoprecipitation with anti-Cdc20 antibody. The extracts (lanes 1–6), chromosomal fractions (lanes 7–12), and Cdc20 immunoprecipitates (lanes 13–18) were immunoblotted for proteins indicated on the left. The band above Mad2 in lanes 13–18 is the IgG light chain. The blots for Mad1 in Cdc20 immunoprecipitates and for Cdc20 in the chromosomal fractions are not shown, because of a lack of specific association. Exogenously added Mad1 and Mad2 tend to nonspecifically associate with Cdc20 immunoprecipitates, resulting in the high level of Mad2 in lane 18.

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