Fig. 3. Inhibition of BMP signalling by Galntl-1. (A-D) Stage 36 embryos that were injected animally at the 8-cell stage with a total of 2 ng of mRNA encoding xGalntl-1, human GALNTL1 (hGalntl-1) or secreted Galntl-1 (sGalntl-1), with the N-terminal transmembrane domain replaced by the Chordin leader peptide. (E) RT-PCR analysis of animal cap explants from embryos injected with 2 ng xGalntl-1 or 40 pg chordin mRNA at stage 20 for neural (pax6, ncam, otx2), anterior (xag), muscle (myf5) and epidermal (cytokeratin) marker genes. (F) Western blot of the supernatant (SN) and cell pellet of dissociated ectodermal cells expressing the xGalntl-1 or sGalntl-1 with a C-terminal HA-TAG. (G) RT-PCR analysis of animal cap explants from embryos microinjected with mRNA encoding human, mouse (m) or secreted forms of Galntl-1. (H) RT-PCR analysis of animal cap explants from embryos microinjected with mRNA encoding xGalntl-1 (1 ng) alone or in combination with mRNA encoding BMP7/OP-1 (400 pg), dnFGFR-4 (400 pg), dnAlk-4 (400 pg) or N-TCF-3 (400 pg). (I) Western blot for Smad1 or phospho-Smad1 using animal cap lysates from uninjected embryos or embryos microinjected with 200 pg Smad1-FLAG mRNA alone or together with 200 pg xGalntl-1 mRNA. (J) Western blot analysis for Smad1 and phospho-Smad1 from transiently transfected Hek293T cells treated for 2 hours with 40 ng/ml recombinant BMP4 protein. (K) Western blot for phospho-Smad1 or Smad1 using lysates from HEK 293T cells transiently transfected with the indicated expression plasmids. In the RT-PCR experiments -actin or myf-5 served as controls for mesoderm contamination; odc as a loading control.