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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 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.