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. 2002 Aug 5;158(3):487–496. doi: 10.1083/jcb.200204048

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Copyright © 2002, The Rockefeller University Press

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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, BubR1^KR, or BubR1^1–742 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), BubR1^KR (lanes 4 and 9), or BubR1^1–742 (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.