Xenbase Image ID: 155606

Figure 4. The Bub1–Mad1 complex promotes APC/CCdc20 inhibition by MCC components.(A) Flow charts of the in vitro reconstitution of Mps1-stimulated APC/C inhibition by MCC components. The incubation times of each reaction step are indicated. All processes were performed at room temperature. Molecules are not drawn to scale. xAPC/C, the APC/C complex isolated from Xenopus egg extract by immunoprecipitation. Ub, ubiquitin. (B) A collection of recombinant proteins used for the in vitro reconstitution. Relevant protein bands were labeled or indicated by arrows. B1, Bub1ΔK; B3, Bub3; M1, Mad1E; M2, Mad2; CB, Cyclin B1; C1, GST-Cdk1; Mono, monomeric Mad2; Di, dimeric Mad2; FL, full-length. (C) The ubiquitination reactions as depicted in (A) were resolved on SDS-PAGE and blotted with the anti-Myc antibody that detected Myc-Securin. The slow-migrating species represented the poly-ubiquitinated forms of Securin. For the reversine sample, the inhibitor was added to the kinase reaction containing Mps1 and Bub1ΔK–Bub3 prior to ATP addition.DOI: http://dx.doi.org/10.7554/eLife.22513.007Figure 4—figure supplement 1. Characterization of monomeric and dimeric Mad2 in the APC/CCdc20 inhibition assay.(A) UV trace of recombinant purified Mad2 fractionated on a Resource Q column. The first peak belongs to the Mad2 monomer (Mad2Mono), whereas the second peak contains the Mad2 dimer (Mad2Di). (B) Spontaneous MCC assembly with Mad2Mono or Mad2Di and the subsequent inhibition of APC/CCdc20. Experiments were performed as depicted in Figure 4A, except that the Bub1 phosphorylation and Bub1–Mad1 binding steps were omitted. Varying amounts of the MCC mixture were added to APC/CCdc20. The ubiquitination reaction mixtures were resolved on SDS-PAGE and blotted with the anti-Myc antibody that detected Myc-Securin. The slow-migrating species represented the poly-ubiquitinated forms of Securin.DOI: http://dx.doi.org/10.7554/eLife.22513.008 Image published in: Ji Z et al. (2017) © 2017, Ji et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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