Fig. 6. Galntl-1 inhibits the binding of ActR-IIB to type I receptors. (A) Immunohistochemical staining of transiently transfected Cos-7 cells for ActR-IIB containing a C-terminal HA-TAG and xGalntl-1 containing a C-terminal FLAG-TAG. (B) Western blot analyses of stage 11 Xenopus animal cap cells expressing HA-tagged ActR-IIB, Alk-3, Alk-4 and Alk-6 and xGalntl-1. (C) Western blot analyses of transiently transfected HEK 293T cells expressing HA-tagged xActR-IIB and xGalntl-1 and incubated overnight in the presence or absence of 2 mM benzyl-GalNAc. (D) Co-immunoprecipitation of HA-tagged Alk-3, Alk-4 and Alk-6 with MYC-tagged ActR-IIB in the presence or absence of FLAG-tagged xGalntl-1 from lysates of transiently transfected HEK 293T cells. (E) Co-immunprecipitation of Alk-3 with Myc-tagged ActR-IIB or FLAG-tagged BMPR-II in the presence or absence of untagged xGalntl-1. The binding of BMPR-II to Alk-3 does not seem to be inhibited by xGAlntl-1. (F) Co-immunprecipitation of Alk-4-HA with ActR-IIB-Myc in the presence of xGalntl-1, sGalntl-1 or xGalnt-6. Only xGalntl-1 interfered with the formation of the heteromeric complex. In all experiments, 10% of the lysates used for co-immunprecipitation was subjected to direct immunprecipitation and the quantity of protein analysed by western blotting for the different epitopes.