Fig. 5. Inhibition of ActR-IIB by Galntl-1. (A) RT-PCR analysis of animal cap explants analysed at stage 20 from embryos microinjected with 400 pg xGalntl-1 mRNA alone or together with 200 pg mRNA encoding human ALK3, mouse Alk6, human BMPR-II or Xenopus ActR-IIB. (B) RT-PCR analysis of stage 20 animal caps microinjected with 400 pg ActR-IIB mRNA either alone or together with 1 ng xGalntl-1 mRNA. The mesodermal marker genes xbra and wnt8 indicated ActR-IIB activity, whereas otx2 and xag indicated xGalntl-1 activity, and xvent2 served as a marker for ventral mesoderm and odc as a loading control. (C) RT-PCR analysis of stage 20 animal caps microinjected with 40 pg ActR-IIB mRNA alone, together with xGalntl-1 morpholinos, and xGalntl-1 morpholinos and 100 pg xGalntl-1 mRNA. (D) Western blot analysis of Smad and phospho-Smad levels from HEK 293T cells transiently transfected with Smad1-FLAG, BMPR-II and xGalntl-1 (left) or Smad2-FLAG, ActR-IIB and xGalntl-1 (right). (E) Western blot analysis of phospho-Smad2 and Smad2 levels from HEK 293T cells transiently transfected with Smad2-FLAG, ActR-IIB and xGalntl-1 and incubated overnight in the presence or absence of 2 mM benzyl-GalNAc. (F) Whole-mount in situ hybridisation for msx1 using stage 13 embryos microinjected with xGalntl-1 morpholinos, 100 pg xActR-IIB mRNA and 100 pg xGalntl-1 mRNA into dorsal-animal blastomeres at the 4- to 8-cell stage.