Fig. 5. Xenopus Tsg functions as a BMP antagonist in the presence of full-length Chordin, but promotes binding of BMP to its receptor in the presence of Chordin fragments. BMP4 was incubated for 1 hour at room temperature with Chordin, CR1, and affinity purified Xenopus Tsg-HA. Subsequently, type I BMP-receptor-Fc fusion protein (R&D Systems) was added, precipitated using protein-A, and analyzed by anti-BMP immunoblot. (A) Xenopus Tsg makes Chordin a better BMP antagonist; the concentration of each component in nM is indicated. (B) Xenopus Tsg restores binding of BMP4 to its cognate receptor in the presence of 20 nM CR1 (lane 3). Note that at high concentrations (100 nM, lane 6) Xenopus Tsg by itself can function as a BMP antagonist in this biochemical assay. (C) The Chd/BMP/Xenopus Tsg ternary complex was digested for 10 hours at room temperature with Xolloid. Lane 3 shows that BMP4 is reactivated and binds to its receptor. (D) Xenopus Tsg does not bind to BMPR-IA. Xenopus Tsg, BMP4 and the BMPRIA-Fc were incubated 1 hour at room temperature before DSS crosslinker was added. The complexes formed were analyzed by anti-BMP4 western blot after protein A immunoprecipitation. The arrow indicates the BMPR-Fc-BMP4 complex. The band at 75 kDa is unspecific as it is also observed in the absence of BMP4 in the reaction (lane 3).
