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. 2014 Sep 26;9(9):e107928. doi: 10.1371/journal.pone.0107928

Figure 5. Ripply can reduce Tbx6 protein level.

Figure 5

(A) Western blotting with proteins recovered from embryos injected with Flag-tagged zebrafish tbx6 (ztbx6-Flag) mRNA and Myc-tagged zebrafish ripply1 (zripply1-Myc) mRNA. Three hundred pg of tbx6 mRNA with or without 300pg of zebrafish ripply1-Myc mRNA were injected into zebrafish eggs at 1 cell stage. (B, C) Similar experiments shown in (A) were performed with 150pg of Flag-tagged mouse Tbx6 (mTbx6-Flag) mRNA and 50pg of mouse Myc-tagged Ripply2 (mRipply2-Myc) mRNA (B) or 120pg of zebrafish ripply1-Myc mRNA (C). Mouse Tbx6 proteins reduced when injected together with mouse Ripply2 mRNA (B) and zebrafish ripply1 (C). (D) Similar experiments shown in (A) were performed with 300pg of zebrafish tbx6-Flag mRNA, and 300pg of wild-type or FPVQ-mutated form of zebrafish ripply1-6Myc mRNA. In FPVQ-mutated form, this 4 amino acid stretch was replaced with four alanines as previously described [26]. (E) Similar experiments shown in (A) were performed with 150pg of mouse Tbx6-Flag mRNA and 50pg of wild-type or FPIQ-mutated form of mouse Ripply2-Myc mRNA. The FPIQ stretch in the mouse Ripply2 exists at the corresponding position to the FPVQ in the zebrafish Ripply1. In FPVQ-mutated form, this 4 amino acid stretch was replaced with four alanines. When co-injected with the mutated forms of zebrafish ripply1 or mouse Ripply2, the reduction of Tbx6 proteins was canceled. (F) Similar experiments shown in (A) were performed with 50pg of mouse Flag-tagged Brachyury (T) mRNA and 50pg of mouse Ripply2-Myc mRNA. Mouse Ripply2 also reduced mouse Brachyury protein level. As internal controls to validate the consistency between injection experiments, 100pg (B, C, E, F) or 200pg (A, D) of GFP mRNA was also injected and its expression was examined.