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. 2010 Jun 21;285(34):26289–26294. doi: 10.1074/jbc.M109.090597

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 6. — XTNIK is an essential and specific enhancer for β-catenin-Tcf-transactivation. A–C, mRNA for Xβ-catenin (25 pg), nuclear β-galactosidase (500 pg), XTNIK-WT-Myc (500 pg), XTNIK-K54R-Myc (500 pg), 5mis-Control-MO1 (Cont-1) (40 ng), XTNIK-MO1 (40 ng), 5mis-Control-MO2 (Cont-2) (40 ng), or XTNIK-MO2 (40 ng) was injected in the indicated combinations into the animal poles of four-cell stage embryos. Twenty animal caps (AC) per group were dissected at stage 9, cultured for 30 min, and harvested for RNA isolation. Relative mRNA expression of Siamois, Xnr3, and chordin was determined by real-time PCR. The statistical significance with respect to n-β-gal mRNA or control MO-injected controls (*, p < 0.05; **, p < 0.01; ***, p < 0.001) was determined using Student's t test. D, -833pSia-Luc plasmid (50 pg) and pRL-TK plasmid (10 pg) were co-injected with Xβ-catenin (25 pg), n-β-gal (500 pg), XTNIK (WT) (500 pg), or human TNIK (WT) (hTNIK-WT, 500 pg) mRNA in the indicated combinations into the animal poles of four-cell stage embryos. Ten animal caps per group were dissected at stage 9, cultured for 30 min, and harvested for the luciferase assay. The statistical significance with respect to the n-β-gal mRNA-injected control (**, p < 0.01) was determined using Student's t test. E, XTNIK-WT-Myc (500 pg) was injected with or without Xβ-catenin (100 pg) mRNA in the ventral marginal zone of embryos. Embryos were harvested at stage 10, and a ChIP assay was performed. F–I, mRNA for Xnr5 (5 pg), BMP4 (500 pg), nuclear β-galactosidase (500 pg), XTNIK-WT-Myc (500 pg), or XTNIK-K54R-Myc (500 pg) and for 5mis-Control-MO1 (Cont-1) (40 ng), XTNIK-MO1 (40 ng), 5mis-Control-MO2 (Cont-2) (40 ng), or XTNIK-MO2 (40 ng) was injected in the indicated combinations into the animal poles of four-cell stage embryos. Twenty animal caps per group were dissected at stage 9, cultured for 1.5 h, and harvested for RNA isolation. Relative mRNA expression of Xnr1, Xbra, Xvent-1, and msx1 was determined by real-time PCR. Bars represent mean ± S.D. J, mRNA for XTNIK-WT-Myc (500 pg) and HA-XTCF4 (500 pg) was injected into the dorsal marginal zone of four-cell stage embryos and harvested at stage 9, and immunoprecipitation was performed.