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. 2010 Aug 23;190(4):501–509. doi: 10.1083/jcb.201001083

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© 2010 Di Fiore and Pines

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 4. — Cyclin A–associated Cks1 increases the efficiency of cyclin A ubiquitylation. (A–D) Cells were injected in G2 phase with cyclin A (1–98)–Venus-Cks1 (A and B) or cyclin A (1–165)–Venus-Cks1 (C and D), and the fluorescence was measured through mitosis. Time is relative to NEBD (A and C) or anaphase (B and D). Error bars indicate mean ± SD of 19 cells from two experiments (A and B) or 24 cells from two experiments (C and D). (E) In vitro ubiquitylation assay of cyclin A (1–165)–Venus (lanes 1–9) or cyclin A (1–165)–Venus-Cks1 (lanes 10–19). Reactions were performed for the indicated time (shown in minutes) before analysis by SDS-PAGE and phosphoimaging. Control reactions are without APC/C (lanes 9 and 18) or E2 (lane 19). (F and G) Quantification of ubiquitin conjugates in E with one to five ubiquitin (F) and more than five ubiquitin (G) molecules were normalized to the total amount of ubiquitylated substrate. (F and G) Error bars indicate mean ± SEM from four experiments. (H) Ubiquitylation reactions for cyclin A (1–165)–Venus (lanes 1–4) and cyclin A (1–165)–Venus-Cks1 (lanes 5–8). 100 times excess of unlabeled cyclin A (1–165)–Venus (lanes 3 and 7) or cyclin A (1–165)–Venus-Cks1 (lanes 4 and 8) was added as a competitor at the beginning of the reaction. (I) Quantification of reactions in H. Numbers correspond to lanes in H. *, unmodified substrate; **, P < 0.01; ***, P < 0.05 (calculated using Student’s t test).