Fig. 5. ARF1 and ARF2 are activated by distinct signalling molecules. (A) Single cell embryos were injected with 200 pg synthetic RNA encoding activin ßA or 1.5 ng RNA encoding derrière, Xnr1, Xnr2,VegT or BMP4. Embryos were cultured until 80 minutes post stage 8 (to detect ARF1, upper panel) or 240 minutes post stage 8 (to detect ARF2, lower panel). Nuclear extracts were prepared and assayed by bandshift assay on the ARE in the presence or absence of anti-Smad2/3 antibody. (B) Whole embryo extracts were prepared from injected embryos at 80 minutes post stage 8 (upper panels) or stage 10.5 (lower panels) and analysed by western blotting with an antibody against activated phosphorylated Smad2 (anti-P-Smad2), or Smad2/3. Xenopus embryos contain no Smad3 at this time (Howell et al., 2001). The upper band is full-length Smad2; the lower band is the spliced isoform missing exon 3 in the MH1 domain (Faure et al., 2000). (C) XFast-3 expression is inhibited by activin, Xnr1 or Xnr2. Total RNA, prepared from injected embryos at stage 10.5, was analysed by RNase protection using probes against XFast-1, XFast-3 or FGFR. Protected fragments are as indicated.