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. 2013 Dec 1;27(23):2531–2536. doi: 10.1101/gad.229195.113

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© 2013 Welcker et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported), as described at http://creativecommons.org/licenses/by-nc/3.0/.

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Figure 3. — Differential regulation of endogenous substrates by endogenous Fbw7 monomers. (A) Ectopic wild-type or dimerization mutants of Flag-Fbw7 expressed in 293A cells were subjected to denaturing and nondenaturing gel analysis and Western-blotted. The positions of Fbw7 monomers and dimers are indicated. (B) Engineered HCT116 cells express monomeric Fbw7. Three independent clones were generated (ΔD, A7, and B6) using two different gene targeting strategies (see the Materials and Methods). Analysis was as in A using immunoprecipitation-Western with Fbw7-specific antibodies. The position of monomers is indicated. (C,D). HCT116 cells expressing Fbw7 dimers (wild type [wt]) or monomers (ΔD) and Fbw7-null cells (−/−) were arrested by aphidicolin in S or G2/M phase or nocodazole in prometaphase or left unsynchronized (asyn) and analyzed for cyclin E abundance and activity. The bar graph depicts the relative amount of cyclin E activity. (E) HCT116 lines (wild type, null, and monomer) were analyzed by quantitative Western blot for c-Myc half-life after cycloheximide chase. The plot averages two independent experiments.