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. 2005 Sep 12;170(6):873–880. doi: 10.1083/jcb.200505040

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Copyright © 2005, The Rockefeller University Press

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Figure 4. — Microtubule capture by CENP-E in vitro produces a ternary complex of microtubule–CENP-E–BubR1 in which CENP-E–activated BubR1 kinase activity is silenced. (A) In vitro kinase assays were performed with combinations of purified GST–BubR1 (lane a), CENP-E (lane b), and 1 μM, 0.1 μM, 10 nM, 1 nM, or 0.1 nM GMPCPP microtubules (lanes 4–8, respectively), 1 μM tubulin (lane 9), or 33 μM GMPCPP (lane 10). (B and C) CENP-E, BubR1, and microtubules form a ternary complex. After centrifugation through a 40% sucrose cushion, BubR1–CENP-E–GMPCPP microtubule complex formation was assayed by (B) immunoblot or (C) triple immunofluorescence for BubR1 (red), CENP-E (green), and microtubules (blue).