Figure 8.

XGef exchange factor activity and the interaction between XGef and CPEB are involved in early CPEB phosphorylation. (A) Summary of the influence of XGef-containing domain mutations on progesterone induced oocyte maturation. (B) His-CPEB WT bound to nickel beads was incubated with extracts prepared from oocytes overexpressing HA-XGef, HA-XGef(Δ65–234), and HA-XGef(65–360) isolated 150 min after progesterone addition, in the presence of [γ-³²P]ATP. Extracts from HA-Globin–overexpressing oocytes selected at prophase I (PI) and 150 min after progesterone addition were analyzed in parallel as a control. The ratio of phosphorylated CPEB (³²P) to His-CPEB (Coomassie stain, CS) is indicated. The levels of endogenous Mos protein and pMAPK in each sample were analyzed by SDS-PAGE and immunoblotting (IB) with anti-Mos and anti-pMAPK antibodies. Anti-PCNA antibodies were used to detect PCNA and confirm equivalent sample loading (bottom). Figure 6A shows the time course of GVBD for these oocytes. (C) Oocytes were injected with affinity-purified XGef antibody (anti-XGef) or nonspecific IgG (ns IgG), and progesterone was added. The percentage of GVBD observed 8 h after progesterone addition is indicated for each treatment in the top histogram (33 oocytes each). An asterisk (^*) indicates that no oocytes underwent GVBD. His-CPEB WT bound to nickel beads was incubated with extracts prepared from ns IgG-(lanes 1 and 2) and anti-XGef antibody (lanes 3 and 4)-injected oocytes isolated at prophase I and 150 min after progesterone addition, in the presence of [γ-³²P]ATP. The autoradiogram (³²P) and CS image of the same gel are shown.