Fig. 5. Inhibitory effects of xTGIF2 on TGFβ and BMP signalings in Xenopus. (A) Partial secondary axis (indicated by ^*) was observed in tadpole stage embryos injected with xTGIF2 (1 ng) mRNA into one ventral vegetal cell. (B) xTGIF2 (1 ng) mRNA and activin (100 pg) mRNA were injected separetely or together into the animal pole of two-cell stage embryos. Animal caps were analyzed at gastrula stage (stage 11) for the expression of indicated markers by RT-PCR. (C) Animal caps injected with xTGIF2 (1 ng) mRNA were analyzed at gastrula stage (stage 11) for the expression of indicated markers by RT-PCR. (D) Luciferase assay with BMP inducible VENT2-luciferase (VENT2-Luc.) reporter construct. Two-cell stage embryos were injected with VENT2-Luc. alone or in combinations with BMP4 (200 pg) and/or xTGIF2 (1 ng) mRNAs, as indicated. Embryos were harvested at the onset of gastrulation and assayed for luciferase activity. (E) Immunoprecipitation (IP) of flag-xTGIF2 and endogenous Smad1 or Smad2. Four-cell stage embryos were injected into the the vegetal pole (ventrally for Smad1 IP; dorsally for Smad2 IP) with flag-xTGIF2 (1 ng), lysates were prepared at gastrula stage (stage 11) and immunoprecipitated with anti-flag antibody and analyzed by immunoblot (IB) with anti-Smad1 and anti-Smad2 antibodies. The expression of flag-xTGIF2, Smad1 and Smad2 was checked by immunoblotting on the crude extracts used for the IP reaction. As loading control, the membranes were stripped and reprobed with anti--tubulin. Chd, chordin; EK, epidermal keratin; xbra, brachyury.