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Fig. 7. XGef is present in a complex with MAPK. (A) Endogenous XGef associates with a U0126 sensitive kinase that can phosphorylate CPEB. Some oocytes were treated with U0126 for 1 hour prior to progesterone addition; protein extracts were prepared from oocytes collected at the indicated times (hr in pg). Endogenous protein complexes were immunoprecipitated with anti-XGef antibody or non-specific IgG. Either recombinant His-CPEB tethered to Ni beads or blank beads alone were added to the immunoprecipitated complexes and incubated in the presence of [-³²P]ATP. Ni-bead-bound samples were analyzed by SDS-PAGE, Coomassie staining and autoradiography. (B) Immunoblot analysis for the samples used in A with anti-P-MAPK and anti-MAPK antibodies. Asterisk indicates 100% GVBD oocytes. (C) Extracts from A were used in an H1 kinase assay. Asterisk indicates 100% GVBD oocytes. (D) MAPK is present in HA-XGef immunoprecipitates. Protein extracts from prophase oocytes (–pg, not exposed to progesterone) and early meiotic oocytes (+pg, exposed to progesterone for 3 hours), expressing HA-XGef or not were used in immunoprecipitations with anti-HA antibody conjugated to Protein-G-agarose. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with anti-MAPK (top panel) and anti-XGef antibodies (middle panel). Input samples were analyzed with MAPK antibody (bottom panel). CS, Coomassie staining; ³²P, autoradiography; IB, immunoblotting; IP, immunoprecipitation.

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