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Fig.S.4 Uch37 specifically regulates Wnt signaling. (a-d) RT-PCR analysis using animal cap explants. Co MO (20ng) or Uch37 MO (20ng) were respectably or co-injected with indicated mRNAs encoding Wnt8 (10pg and 20pg), BMP4 (50pg and 100pg), Xnr1 (50pg and 100pg), or eFGF (100pg and 200pg) in animal blastomeres of four-cell stage embryo. Animal cap explants were dissected at stage 9 and cultured until stage 11. siamois, Wnt-target gene (a); Sizzled, Bmp4-target gene (b); Xbra, target gene of Xnr1 (c) and eFGF (d). (e) Short hairpin RNA (shRNA) of Uch37 efficiently reduced levels of Uch37 protein in HepG2 cells. (f) TOPflash assay in HepG2 cells. Reporter constructs were transfected alone or co-transfected with constitutively active LRP6 (LRP6∆N) into stable cells (sh Control and sh Uch37). 48h after transfection, luciferase activity was measured. (g) qPCR analysis for the expression of Wnt-target genes (c-myc and cyclinD1) in stable HepG2 cells (sh Control or sh Uch37) treated with or without LiCl (25mM). The quantities of indicated mRNA were normalized by β-actin. Data represent average values from three independent experiments performed. Error bars indicate standard deviations of triplicate. *, p <0.005; **, p< 0.001 (two-tailed Student’s ttest).
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