Figure 4.

xTsg binds to BMP specifically and directly. a, Sequence similarities between Chordin cysteine-rich modules and the N-terminal domain of xTsg. b, Western blot analyses of BMP-4 (1.0 nM) bound to xTsg (1.0 nM) after immunoprecipitation by xTsg in the presence or absence of a 10-fold excess of various growth factors. The control (co) lanes in this and Figs 5-7 contain conditioned medium from mock-transfected 293T cells. To measure the recovery of xTsg after immunoprecipitation, the same membrane was stripped and probed for xTsg protein (bottom). c, Crosslinking analysis of xTsg-BMP-4 complexes with DSS. Left, immunoblot probed for xTsg. Right, the same membrane probed for BMP-4 after stripping. d, Equilibrium binding of increasing concentrations of BMP-4 (0.2-30 nM) to 1 nM Tsg. Binding was for 3 h at 4 °C, and bound and free BMP-4 were separated by immunoprecipitation; crosslinking agents were not used. The amount of bound BMP-4 was determined as described¹². Each data point was in duplicate, and two independent experiments were performed for each protein. Scatchard analyses of the dTsg data gave an apparent K_d of 2.5 nM. The same results and K_d were obtained for the xTsg protein as illustrated by the anti-BMP-4 immunoblot (inset and data not shown).