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Fig. 5. CPEB is phosphorylated by endogenous MAPK during early meiosis. (A) Protein extracts from oocytes collected at the indicated times were used in IP assays with either anti-P-MAPK antibody or non-specific rabbit antibody (NS). His-CPEB tethered to Ni beads were added to the immunoprecipitates and incubated in the presence of [
-32P]ATP. Ni-bead bound samples were analyzed by SDS-PAGE, Coomassie staining and autoradiography. Asterisks indicate 100% GVBD oocytes. (B) Bead-bound samples from A were immunoblotted with anti-MAPK antibody to confirm efficiency of immunoprecipitation. Asterisks indicate 100% GVBD oocytes. (C) In vitro phosphorylation of His-CPEB using recombinant MAPK. His-CPEB or BSA were incubated with the indicated units of MAPK, together with [-32P]ATP. Samples were analyzed by SDS-PAGE, Coomassie staining (CS) and autoradiography (32P). (D) Schema of His-CPEB protein showing positions of MAPK phosphorylation sites determined by LC-MS/MS phosphopeptide mapping after using either recombinant MAPK from experiments described in C or staged oocyte extracts (bottom three lines) as the kinase source. RRM, RNA-recognition motif; ZF, zinc-finger motif; *, S174; PEST, degradation-targeting domain rich in proline, glutamate, serine and threonine. (E) Table of average charge-to-mass values for ionized peptides (b and y) generated after secondary fragmentation of the CPEB tryptic peptide LDSR. (Left) His-CPEB phosphorylated in vitro using MAPK (as shown in D). (Right) His-CPEB phosphorylated by incubation with extracts from early-maturing oocytes. A difference of 80 in the mass-to-charge values of the S174 phosphorylated versus non-phosphorylated peptide ions indicates the presence of the phosphate group. *, S174; CS, Coomassie staining; 32P, autoradiography; IB, immunoblotting; IP, immunoprecipitation.
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