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The Role of 14-3-3ε Interaction with Phosphorylated Cdc25B at Its Ser321 in the Release of the Mouse Oocyte from Prophase I Arrest

Figure 3

Ser321 of Cdc25B is the sole site responsible for 14-3-3ε binding.

Effects of microinjection with HA-tagged-14-3-3ε mRNA solely or co-injection with MYC-tagged-Cdc25B-WT mRNA, MYC-tagged-Cdc25B-Ser321A mRNA or MYC-tagged-Cdc25B-Ser321D mRNA and HA-tagged- 14-3-3ε mRNA on meiotic resumption of mouse oocytes. Experiments were performed in the continued presence of dbcAMP(A–G). HEK293 cells were transfected with 14-3-3ε together with empty vector, Cdc25B-WT , Cdc25B-Ser321A or Cdc25B-Ser321D (H). (A) at the indicated times, the percentages of germinal vesicle breakdown (GVBD) or MII or death were counted in cultured mouse oocytes after various mRNAs microinjection. GVBD, 3 h; MII or death, 20 h. The total number of oocytes undergoing meiotic maturation or death is given on top of bar graph from three independent experiments. (B) the GVBD rate at indicated time points in cultured mouse oocytes after various mRNAs microinjection. (C) H1 kinase activity in oocytes injected with various mRNAs. Each value was expressed as mean±S.D of at least thee independent experiments. (D) Protein extracts from oocytes microinjected with various mRNAs were incubated with histone H1 and [γ−32P] ATP. The protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and incorporation of 32P into histone H1 was visualized by autoradiography. (E) Western analysis of phosphorylation status of Cdc2-Tyr15. GV oocytes injected with various mRNAs were collected 2 h after microinjection. The collected oocytes were immunoblotted with anti-pTyr15 of Cdc2 antibody. (F) Western analysis of Cdc25B-MYC and endogenous Cdc25B expression. The oocytes co-injected with Cdc25B-WT mRNA, Cdc25B-Ser321A mRNA or Cdc25B-Ser321D mRNA and 14-3-3ε mRNA were collected 3 h after injection and their proteins were immunoblotted with anti-myc, anti-Cdc25B or anti-beta-actin antibody. Bars represent means±S.D of thee independent experiments. (G) Western analysis of HA-14-3-3ε and endogenous 14-3-3ε expression. The oocytes injected with 14-3-3ε mRNA solely or co-injected with Cdc25B-WT mRNA, Cdc25B-Ser321A mRNA or Cdc25B-Ser321D mRNA and 14-3-3ε mRNA were collected 3 h after injection and their proteins were immunoblotted with anti-HA, anti-14-3-3ε or anti-beta-actin antibody. Bars represent means±S.D of thee independent experiments. (H) Western blot analysis of Cdc25B interaction with 14-3-3ε (IP) and Western blot analysis demonstrating the expression of HA-14-3-3ε and EGFP-Cdc25B in the cell lysates used to immunoprecipitate Cdc25B protein shown in (IP), an anti-GAPDH antibody was used to determine equal loading of the gel.

Figure 3

doi: https://doi.org/10.1371/journal.pone.0053633.g003