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Fig. 3. Early phosphorylation of CPEB occurs in the absence of Aur-A activity. (A) His-CPEB phosphorylation assays using Aur-A immunodepleted extracts. Protein extracts from oocytes collected after incubation in progesterone (hr in pg) for the indicated times (in hours) were immunodepleted with non-specific IgG antibody, antibody against total Aur-A or no antibody and used in His-CPEB phosphorylation assays. Immunodepleted samples were analyzed by SDS-PAGE and immunoblotted with anti-Aur-A antibody. Phosphorylated His-CPEB signal intensity was adjusted relative to the quantity of input His-CPEB. Asterisk indicates 100% GVBD oocytes. (B) Samples from A were immunoblotted with antibodies against Aur-A, phosphorylated (active) MAPK (P-MAPK), and unphosphorylated and phosphorylated Cdc2. Asterisk indicates 100% GVBD oocytes. (C) The Aurora kinase inhibitor ZM447439 blocks Aur-A activity in vitro. Increasing amounts of recombinant Aur-A were treated with either ZM447439 or DMSO, then incubated with myelin basic protein (MBP) as a substrate in the presence of [
-32P]ATP. (D) ZM447439 inhibits endogenous AurA kinase. Affinity-purified Aur-A antibody or NS-IgG immunoprecipitates from GVBD extracts were incubated with MBP and [-32P]ATP in the presence of ZM447439 or DMSO. (E) ZM447439 does not block CPEB phosphorylation. Protein extracts from oocytes collected at the indicated times were treated with either 20uM ZM447439 or DMSO and used in His-CPEB phosphorylation assays. Asterisk indicates 100% GVBD oocytes. Input extracts were incubated with histone H1 and [-32P]ATP in an H1 kinase assay (pH1, lower panel). (F) ZM447439 does not inhibit GVBD. Oocytes were injected with either 40 nl of 500 µM ZM447439 or DMSO two hours prior to the addition of progesterone. After overnight incubation, the number of oocytes that had achieved GVBD was scored. CS, Coomassie staining; 32P, autoradiography; IB, immunoblotting.
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