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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 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.