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. 2011 Jul 1;22(13):2157–2164. doi: 10.1091/mbc.E11-01-0008

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© 2011 Yamamoto et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Regulated binding of PP2A/B55 to Greatwall. (A) Greatwall releases PP2A/B55 in M phase. Oocytes were injected with mRNA encoding FLAG-Gwl and incubated overnight as described in Materials and Methods. Some oocytes were then treated with progesterone and monitored for entry into meiosis I (MI, GVBD). Extracts from the control (uninjected), G2, and GVBD oocytes were immunoprecipitated with anti-FLAG antibody beads, and the immunoprecipitates were Western blotted for FLAG and the B55 subunit of PP2A. C, control; WT, wild type. (B) Association of Greatwall with PP2A in G2 phase. Oocytes were injected with mRNA encoding FLAG-WT or T748V Gwl and incubated overnight. Some oocytes were then treated with progesterone and monitored for white-spot formation (GVBD, MI). Lysates from the oocytes were precipitated with microcystin–Sepharose beads (MC), and the precipitates were Western blotted for FLAG and for the catalytic subunit of PP2A (bottom). Lysates for Western blotting were also analyzed before immunoprecipitation (top). (C) Kinase-dead Greatwall releases PP2A in M phase. An experiment like that in B was performed, except that G41S Gwl was analyzed in MI (GVBD) oocytes. (D) The C-terminal region of Greatwall binds PP2A in G2. mRNA encoding FLAG-tagged N-terminal (NT) or C-terminal (CT) Gwl was injected into oocytes, followed by overnight incubation. Oocyte lysates were immunoprecipitated with anti-FLAG antibody beads, and the precipitate was Western blotted with FLAG and B55 antibodies, as indicated. (E) The C-terminal region of Greatwall releases PP2A in M phase. Oocytes were injected with CT Gwl mRNA as in D, except that some were treated with progesterone to stimulate entry into meiosis I (MI), as indicated. At GVBD, FLAG-Gwl was immunoprecipitated on anti-FLAG beads from progesterone-treated (MI) or nontreated (G2) oocytes, and the immunoprecipitates were Western blotted for FLAG and B55.