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. Author manuscript; available in PMC: 2015 Jan 23.
Published in final edited form as: Mol Cell. 2014 Jan 23;53(2):179–192. doi: 10.1016/j.molcel.2013.12.022

Figure 2. Microtubules prevent ubiquitylation of spindle assembly factors by the APC/C.

Figure 2

A. Microtubules inhibit ubiquitylation of HURP by APC/CCdc20. 35S-labeled HURP was synthesized in vitro, purified, incubated with RanQ69L and subjected to ubiquitylation by APC/CCdc20. As indicated, HURP was pre-bound to microtubules. Reactions were monitored by autoradiography. B. Microtubules inhibit ubiquitylation of 35S-labeled HURP by APC/CCdh1 isolated from HeLa cells synchronized in G1. Reactions were analyzed in the presence of microtubules as described. C. Microtubules prevent the ubiquitylation of bacterially expressed HURP. HURP1-280, which contains all degrons and microtubule-binding domains, was incubated with microtubules and subjected to APC/C-dependent ubiquitylation. Reactions were analyzed by Western. The asterisks mark crossreactive bands. D. Microtubules do not affect the ubiquitylation of 35S-labeled cyclin B1 cyclin B1 by APC/CCdc20. See also Figure S1.