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Fig. 1. Blocking Rho-family GTPase function with Clostridium difficile Toxin B does not interfere with CPEB phosphorylation during early meiosis. Toxin-B-injected oocytes were incubated for 3 hours prior to stimulation of meiosis with progesterone. PD, pulldown; IB, immunoblotting. (A) Protein extracts from oocytes collected post injection or after incubation in progesterone (hr post injection or hr in pg, respectively) at the indicated times (in hours) were treated to activate GTPases and then incubated with GST-PAK-PBD or GST-Rhotekin-RBD. Samples bound to glutathione-beads were analyzed by immunoblotting with anti-Cdc42, anti-Rac1, anti-RhoA and anti-GST antibodies, as indicated. The bottom panel is a representative immunoblot with anti-GST antibody to confirm equivalent loading of GST-PAK-PBD. Similar results were obtained with beads loaded with GST-Rhotekin-RBD. (B) Extracts from parallel sets of oocytes were incubated with His-CPEB and [-³²P]ATP in an in vitro His-CPEB phosphorylation assay. Ni-bead bound samples were analyzed by SDS-PAGE, Coomassie staining (CS) and autoradiography (³²P). (C) Progression to GVBD, monitored by appearance of a white spot at the animal pole, was followed in Toxin-B- and control-injected oocytes, and plotted versus incubation time.

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