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. 2008 Jul 14;182(1):19–26. doi: 10.1083/jcb.200711040

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© 2008 Bonetti et al.

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.jcb.org/misc/terms.shtml). 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 2. — NPM is required for Fbw7γ activity. (a) WB analysis in p53−/− and dKO MEFs infected with Myc-ER and, where indicated (+), HA-Fbw7γ–expressing retroviruses. Cells were treated with 4-OHT for the indicated hours or left untreated (0 h). (b) QPCR of anti–c-Myc ChIP on target promoters (as indicated) after 4-OHT treatment of the same cells as in panel a. (c) WB analysis in p53−/− and dKO MEFs infected with retroviruses expressing HA-Fbw7γ (+) or control retroviruses (−) as indicated. c-Myc levels have been calculated by densimetric analysis, normalizing band intensity to actin and control p53−/− cells. (d) QPCR analysis of anti c-Myc ChIP on target promoters (as indicated) of the same cells as in panel c. (e) dKO MEFs were cotransfected with plasmids expressing NPM1 and flag-Fbw7γ (left), flag-Fbw7α (middle), and flag-Fbw7β (right). Total lysates and immunoprecipitates were blotted with antibodies against NPM (NPMa) or anti-flag. (f) GST pull-down assay using in vitro translated ³⁵S-labeled Fbw7γ or Fbw7α and equal amounts of GST or GST-NPM proteins. Data represent the mean of three determinations ± SEM.