Fig. 4. Biochemical analyses of xTsg mutant proteins. (A) RT-PCR of animal cap explants injected with 2 ng xTsg, xTsg^W67G or xTsg^C198A mRNA. (B) Immunoprecipitation of BMP4 bound to xTsg. Recombinant BMP4 (5 nM) was preincubated with either 20 nM xTsg^W67G, xTsg^C59A or wild-type xTsg protein (lanes 2, 3, 4). Immunoprecipitation was performed with a polyclonal antibody recognizing an N-terminal peptide present in the Tsg expression vector (Piccolo et al., 1999) and the immunoblot probed with anti-BMP4 or anti-Flag antibodies. (C) Tsg-Chordin complexes formed after crosslinking of 20 nM of the indicated affinity-purified xTsg proteins to 5 nM xChordin protein with DSS (disuccinimidyl suberate). xTsg proteins were visualized via the FLAG epitope. Note that xTsg^W67G binds to Chordin (lane 4) and that xTsg^C198A does not (lane 6). (D) Chemical crosslinking of BMP-Chordin-Tsg ternary complexes after incubation of BMP4 (5 nM, lane 1) with Chordin (5 nM, lane 2) and xTsg (20 nM, lane 3) or xTsg^W67G (20 nM, lane 4) proteins. The western blot was stained with anti-BMP4 monoclonal antibody. (E) Anti-FLAG immunoblot of the same experiment shown in D, after stripping of the BMP4 antibody and incubation with an anti-FLAG antibody. (F) Crosslinking with DSS of 293T supernatants from cell cultures transfected separately with the indicated murine expression vectors and then co-cultured. Formation of a ternary BMP4-Chordin-Tsg complex led to a significant increase in BMP avidity by the monoclonal antibody (lane 4). The amounts of BMP4 in the supernatants vary, due to binding to the extracellular matrix of the cultured cells.