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Fig. 9. BubR1 is recruited to phosphorylated but not to unphosphorylated kinetochores. All cells were initially lysed in the absence of microcystin to become dephosphorylated and, after different treatments, fixed and stained for 3F3/2, BubR1, CID and DNA (blue). (A) Cells treated with ATP in the absence of microcystin (ATP-Mc) retain normal CID staining but BubR1 and 3F3/2 labelling is abolished. (B) Cells treated with ATP plus microcystin (ATP+Mc) show CID and 3F3/2 staining but endogenous BubR1 is depleted during extraction. (C) Cells rephosphorylated with ATP but without microcystin and subsequently incubated with S2 mitotic extract (M ext). Note that 3F3/2 epitopes are not rephosphorylated and that exogenous BubR1 is not recruited to kinetochores. (D) Cells rephosphorylated in the presence of microcystin and subsequently incubated with S2 mitotic extract show rephosphorylated 3F3/2 epitopes and strong accumulation of BubR1 at kinetochores. (E) Cells rephosphorylated in the presence of microcystin and incubated with an S2 mitotic extract mostly depleted of BubR1, show strongly labelled 3F3/2 phosphoepitopes but only weak accumulation of BubR1. Scale bars: 5 µm.
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