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. Author manuscript; available in PMC: 2011 Apr 4.
Published in final edited form as: Cell. 1996 Aug 23;86(4):589–598. doi: 10.1016/s0092-8674(00)80132-4

Figure 3. Chordin Binds to BMPs But Not to Activin.

Figure 3

Immunoprecipitation (A and B) and cross-linking (C) results were detected by nonreducing SDS–polyacrylamide gel electrophoresis and Western blot using an anti–BMP-4 monoclonal, as described in Experimental Procedures.

(A) Immunoprecipitation with anti–NH2-Chd antibody of a mixture of 0.25 nM Chd and 0.5 nM BMP-4. Lane 1: control protein purified from an irrelevant viral infection serves as negative control. Lane 2: BMP-4 is bound to chordin and visualized in Western blots probed with anti–BMP-4 monoclonal antibody. Lanes 3–5: BMP-4 binding cannot be competed by 5 nM of the unrelated cytokines platelet-derived growth factor-α, insulin-like growth factor I, and epidermal growth factor.

(B) The immunoprecipitation assay was used to compete the binding of BMP-4 (lane 1) with increasing amounts of BMP-2 (lanes 2–5) and a 10-fold excess of activin or TGF-β1 (lanes 6 and 7). Binding revealed by the anti–BMP-4 monoclonal is shown in the top row; after stripping with β-mercaptoethanol and SDS, the same filter was probed with anti–BMP-2 monoclonal and is shown in the bottom row. Note that BMP-2 (but not activin or TGF-β1) competes with BMP-4 for binding to chd, reaching saturation at 2.5 nM.

(C) Cross-linking analysis of Chd–BMP-4 complexes using dithiobis-(succinimidyl)propionate. Lanes 1 and 2: BMP-4 alone and Chd alone. Chd and BMP-4 form a complex of 150 kDa (lane 3) which is competed by a 10-fold excess of BMP-2 but not of activin (lanes 4 and 5). Control protein extracts from an irrelevant baculovirus infection show no binding (lane 6). We note that BMP-4 may undergo a conformational change when bound to chordin. This is indicated by the observation that the Chd–BMP complex is recognized more intensely than unbound BMP-4 by the monoclonal antibody (compare lanes 1 and 3). The simplest interpretation for this observation is that the monoclonal epitope becomes more accessible in the Chd–BMP-4 complex. This increase in immunostaining was even more evident when BMP-4/7 heterodimer, which per se was barely detected by the monoclonal antibody, was bound by chordin (data not shown).