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).