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Fig. 1. Tsg and Bmp7 interact biochemically and are expressed in overlapping domains. (A) Bmp7 co-immunoprecipitates with Tsg. Top panel was probed with an anti-Bmp7 antibody. Bmp7 was pulled down only in the presence of Tsg (compare lanes 1 and 2). This Tsg/Bmp7 interaction was not competed by a sixfold excess of TGFß2 (lane 3) but was partially competed by equimolar amounts of Bmp4 (lane 4). Middle panel provides a control for the amount of Tsg immunoprecipitated; the bottom panel serves as a loading control for antibodies on the beads. (A') Crosslinking of Tsg and Bmp7 proteins by DSS. A bimolecular complex of Tsg and Bmp7 is formed in lane 3. Tsg protein is HA tagged. Top panel (crosslinked) and middle panel (no crosslinker) were probed with an anti-HA antibody; bottom panel (no crosslinker) was probed with anti-Bmp7 antibody. Tsg protein forms a homodimer when crosslinked (lane 1, top panel). In the presence of Bmp7 (lane 3, top panel) a bimolecular complex (consistent with a homodimer of Tsg binding a homodimer of Bmp7) runs around 110 kDa. (B-D') Expression of Tsg and Bmp7 analyzed by whole-mount in situ hybridization and ß-gal staining. (B,B') Bmp7 and Tsg are co-expressed in allantois (al), axial and lateral mesoderm (am) and gut endoderm (en) at 8.25 dpc. Tsg expression is visualized via an in-frame fusion to lacZ. (C,C') At 9.0 dpc, Tsg and Bmp7 are found in gut endoderm, branchial arch one (a1), eye vesicle (ev) and otic vesicle (ov). (D,D') At 10.5 dpc, Tsg and Bmp7 are co-expressed in posterior ventral mesoderm (vm), limb bud (lb), first pharyngeal arch and basal telencephalon (bt). (E,F) Sections of 8.25 and 9.0 dpc embryos showing Tsg mRNA in endoderm, neurectoderm, head mesenchyme (hm) and ventral mesoderm (vm). (G) Section of a 9.0 dpc embryo showing expression of Bmp7 in ventral mesoderm and endoderm of the hindgut. Plane of sections are indicated in B',C,C'. h, heart; hg, hindgut; me, mesoderm; md, mandibular component of first pharyngeal arch; mx, maxillary component of first pharyngeal arch; ne, neuroectoderm; no, node.