Fig. 4. Binding of Xenopus Tsg to Chordin requires an uncleaved C-terminal Xolloid cleavage site. (A) Schematic representation of the cleavage sites of Xolloid in Chordin (arrows). Fragments of chordin mimicking the products of Xolloid digestion were prepared in 293T cells and designated as Chd-A, Chd-B, Chd-C, Chd-A+B and Chd-B+C. Each of these constructs contains the Xenopus Chordin signal peptide and an N-terminal Flag tag (except for Chd-A+B, which lacks the flag tag). (B) Western blot analysis of Xenopus Tsg-HA (5 nM) bound to the different Chordin fragments (5 nM each) after immunoprecipitation of Chordin in the absence (lanes 1 to 4) or presence (lanes 5 to 8) of 5 nM BMP4. As a loading control, membranes were stripped and probed with anti-Flag (lanes 1 to 8) or anti-I-Chd (lanes 9 to 11). (C) Anti-BMP4 immunoblot analysis of Xenopus Tsg-BMP-Chd ternary complexes after crosslinking with DSS (disuccinimidyl suberate). (D) BMP4 is dislodged from preformed Chd-A/BMP4 complexes by Xenopus Tsg. Chd-A and BMP4 were incubated for 1 hour at room temperature, followed by another hour of incubation in the presence of increasing amounts of Xenopus Tsg, and after that the crosslinker DSS was added. The complexes formed were analyzed by anti-BMP4 immunoblot. Note that Xenopus Tsg dislodges most the BMP4 from Chd-A at equimolar concentrations (lane 5) and that no Chd-A/BMP4/Xenopus Tsg ternary complexes were formed at any concentration.
