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. 2008 Jul 14;182(1):27–33. doi: 10.1083/jcb.200712080

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© 2008 Tan 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 4. — Binding to PP1 is essential for Mars function. (A) Mars binds to PP1α87B in a GST pulldown (PD) assay. Binding requires Phe839 of Mars. arm-GAL4 UAS-HA-PP1α87B fly extracts were incubated with lysates from Escherichia coli expressing GST-Mars^WT or Mars^F839A. Mars complexes were purified on glutathione-Sepharose and immunoblotted with HA to test for coprecipitation of PP1α87B. Blots of total extracts confirmed equal levels of HA-PP1 and GST-Mars in these experiments. (B) Mars coprecipitates PP1 from D. melanogaster embryonic nuclear extracts. arm-GAL4 (−) and arm-GAL4 UASP-FM-mars (+) embryo extracts were subjected to immunoprecipitation (IP) with Myc antibodies followed by immunoblotting with PP1 antibodies. Blots of total extracts confirmed levels of PP1 and FM-tagged Mars. (C) HURP coprecipitates PP1 from HeLa cell nuclear extracts. Immunoprecipitation of extracts with rabbit HURP antibody or with rabbit random IgG (Rb IgG) was followed by immunoblotting with PP1 antibodies to test for binding. (D) Hatch ratios, plotted as the mean ± standard error of seven experiments. Loss of one copy of PP1α87B enhances mars^1/+ to semilethality. Ectopic mars^FA fails to rescue lethality of embryos laid by mars mutant mothers. P-values indicated by an asterisk are not statistically significant. (E) Ectopic Mars^WT and Mars^F839A have identical distributions on mitotic spindles. Fixed embryos from arm>mars^WT or arm>mars^F839A mothers were stained to reveal the distribution of α-tubulin (green), FLAG-tagged Mars (red and greyscale), and DNA (blue) during mitosis. M, metaphase; T, telophase.