Journal: The Journal of Biological Chemistry

Article Title: The 14-3-3γ isoform binds to and regulates the localization of endoplasmic reticulum (ER) membrane protein TMCC3 for the reticular network of the ER

doi: 10.1016/j.jbc.2022.102813

Figure Lengend Snippet: Phosphorylated serine 15 in the deduced 14-3-3 binding motif is required for potent binding to 14-3-3γ . A , the N-terminal region of TMCC3 highlighting the deduced 14-3-3 binding motifs. Serine residues being the possible phosphorylation sites in three deduced 14-3-3 binding motifs are indicated in red . Arginine, serine, and proline residues at −3, 0, and +2 positions, respectively, in the deduced 14-3-3 binding motif predicted with the highest score are underlined. B , serine 15 is required for potent binding to 14-3-3γ. Indicated combinations of HA-TMCC3-D1, HA-TMCC3-D1-S15A, HA-TMCC3-D1-S15/25/46A, and Flag-14-3-3γ were transfected into HEK293 cells, followed by immunoprecipitation with the anti-Flag mAb. The samples were immunoblotted with the anti-HA mAb and the anti-Flag pAb. The arrowheads indicate the bands of interest. The asterisk s indicate the nonspecific bands from the light chain of IgG. C , serine 15 is phosphorylated. HA-TMCC3-D1, HA-TMCC3-D1-S15A, and HA-TMCC3-D1-S15/25/46A were transfected into HEK293 cells, followed by immunoprecipitation with the anti-HA mAb. The samples were immunoblotted with the anti-phospho-14-3-3 binding motif pAb and the anti-HA pAb. The asterisk indicates the nonspecific bands from the light chain of IgG. TMCC3, transmembrane and coiled-coil domain family 3.

Article Snippet: The antibodies were purchased from the following commercial sources: mouse anti-HA mAb (BioLegend; Cat. No. 901514), rabbit anti-HA pAb (Sigma-Aldrich; Cat. No. H6908), rat anti-HA mAb (Roche; Cat. No. 11867423001), mouse anti-FLAG mAb (Sigma-Aldrich; Cat. No. F1804), rabbit anti-FLAG pAb (Sigma-Aldrich; Cat. No. F7425), rabbit anti-TMCC3 pAb (Sigma-Aldrich; Cat. No. HPA014272), rabbit anti-CLIMP-63 pAb (Bethyl Laboratories; Cat. No. A302–257A), mouse anti-α-tubulin mAb (Sigma-Aldrich; Cat. No. T6199), mouse anti-14-3-3 mAb (Santa Cruz Biotechnology; Cat. No. sc-1657), rabbit anti-phospho 14-3-3 binding motif pAb (Cell Signaling; Cat. No. 9601), mouse anti-PDI mAb (Abcam; Cat. No. ab2792), rat anti-GFP mAb (Nacalai; Cat. No. 04404–84), rabbit anti-GFP pAb (MBL; Cat. No. 598), and mouse anti-GAPDH mAb-HRP-DirecT (MBL; Cat. No. M171–7).

Techniques: Binding Assay, Transfection, Immunoprecipitation

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0053633

Figure Lengend Snippet: (A) the mRNA levels of 14-3-3 isoforms at GV stage of mouse oocytes. mRNA of 120 mouse oocytes of GV stage were extracted (described in the“ ” Section). RT-PCR products using primers for specific 14-3-3 isoforms are observed in ethidium bromide-stained agarose gel. ε, β, γ, η, σ, τ and ζ represent seven 14-3-3 isoforms. (B) the mRNA levels of 14-3-3ε at GV and GVBD stage of mouse oocytes. Line GV: mouse oocytes at GV stage. Line GVBD: mouse oocytes at GVBD stage. (C) A protein band of approximately 28 kDa from 200 mouse oocytes at GV or GVBD stage is detectable by Western blot with an anti-14-3-3ε antibody (upper panel). In contrast an anti-β-actin antibody detected a low molecular weight band of approximately 43 kDa (lower panel). (D) Western blot analysis 14-3-3β protein expression with an anti-14-3-3β antibody from 300 mouse oocytes at GV or GVBD stage, A protein band of approximately 29 kDa (upper panel). Line Brain: mouse brain protein as positive control. Shown is a representative of three independent experiments.

Article Snippet: The blots were probed overnight at 4°C in 1% milk in TBST with the following antibodies: rabbit anti-14-3-3ε (1∶1000) (Abcam); rabbit anti-14-3-3β (1∶800) (cellsignaling); rabbit anti-Myc (1∶1000) (Clontech); rabbit anti-HA (1∶800) (Sigma); Tyr(P)15 of Cdc2 (1∶500; Santa Cruz Biotechnology) and rabbit anti-β-actin (1∶400) (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Western Blot, Molecular Weight, Expressing, Positive Control

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0053633

Figure Lengend Snippet: (A) 120 GV-oocytes microinjected with 14-3-3ε siRNA or control siRNA (5pl of 20 µM) were collected 24 h after microinjection. Oocytes microinjected with control siRNA and no injection served as control. mRNA of 14-3-3ε was detected by RT-PCR using primers specific for 14-3-3ε. (B) Protein expression of 14-3-3ε was examined by western blot with an anti-14-3-3ε antibody. β-actin used as endogenous internal reference demonstrate 14-3-3ε siRNA specificity and equal loading. (C) at the indicated times, the percentages of germinal vesicle breakdown (GVBD) or MII or death were counted in cultured mouse oocytes after 14-3-3ε siRNA or control siRNA microinjection. GVBD, 24 h; MII or death, 30 h. The total number of oocytes undergoing meiotic maturation or death is given on top of bar graph from three independent experiments. (D) H1 kinase activity in oocytes injected with 14-3-3ε siRNA or control siRNA or no injection groups. Each value was expressed as mean±S.D of at least three independent experiments. (E) Protein extracts from oocytes microinjected with 14-3-3ε siRNA or control siRNA and no injection groups were incubated with histone H1 and [γ -32 P] ATP. The protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and incorporation of 32 P into histone H1 was visualized by autoradiography. (F) Western analysis of phosphorylation status of Cdc2-Tyr15. GV oocytes injected with 14-3-3ε siRNA or control siRNA were collected 24 h after microinjection. The collected oocytes were immunoblotted with anti-pTyr15 of Cdc2 antibody. (G) Co-localization of Endogenous Cdc25B and 14-3-3ε in mouse oocytes injected with 14-3-3ε siRNA or control siRNA. red fluorescent Cdc25B signals and green fluorescent 14-3-3ε signals were co-localized in the cytoplasm in oocytes injected with control siRNA, while red fluorescent Cdc25B signals were translocated from the cytoplasm to the nucleus in oocytes injected with14-3-3ε siRNA. Scale bar = 20 µm.

Article Snippet: The blots were probed overnight at 4°C in 1% milk in TBST with the following antibodies: rabbit anti-14-3-3ε (1∶1000) (Abcam); rabbit anti-14-3-3β (1∶800) (cellsignaling); rabbit anti-Myc (1∶1000) (Clontech); rabbit anti-HA (1∶800) (Sigma); Tyr(P)15 of Cdc2 (1∶500; Santa Cruz Biotechnology) and rabbit anti-β-actin (1∶400) (Santa Cruz Biotechnology).

Techniques: Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Cell Culture, Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Autoradiography

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0053633

Figure Lengend Snippet: 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 −32 P] ATP. The protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and incorporation of 32 P 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.

Article Snippet: The blots were probed overnight at 4°C in 1% milk in TBST with the following antibodies: rabbit anti-14-3-3ε (1∶1000) (Abcam); rabbit anti-14-3-3β (1∶800) (cellsignaling); rabbit anti-Myc (1∶1000) (Clontech); rabbit anti-HA (1∶800) (Sigma); Tyr(P)15 of Cdc2 (1∶500; Santa Cruz Biotechnology) and rabbit anti-β-actin (1∶400) (Santa Cruz Biotechnology).

Techniques: Injection, Transfection, Plasmid Preparation, Cell Culture, Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Autoradiography, Western Blot, Expressing