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Fig. 8. BubR1 and Bub3 loss from kinetochores is specifically due to 3F3/2 epitope dephosphorylation. Condensed chromosomes were purified from S2 colchicine-arrested cells in the presence of microcystin to maintain kinetochore phosphoepitopes. In all preparations DNA was stained with DAPI (blue). (A,E) Isolated chromosomes were fixed and immunostained with 3F3/2, anti-BubR1 and anti-Bub3 antibodies. Note the strong kinetochore labelling for all antibodies. (B,F) Isolated chromosomes were incubated with lambda phosphatase ({lambda}PPase) leading to complete dephosphorylation of the 3F3/2 epitopes and to loss of BubR1 and Bub3 from kinetochores. (C,G) Chromosomes incubated with lambda phosphatase buffer alone ({lambda} buffer) retain 3F3/2 phosphoepitopes, as well as BubR1 and Bub3 proteins. (D,H) Isolated chromosomes were first incubated with the 3F3/2 antibody and then treated with lambda phosphatase before immunostaining for BubR1 or Bub3. Preincubation with 3F3/2 antibodies blocks the accessibility of the phosphatase to the 3F3/2 phosphoepitopes causing BubR1 and Bub3 to remain at kinetochores. Scale bar: 5 µm.





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