Fig. 10. Derrière physically associates with other members of the TGFß superfamily, including Xnr2 and BMP4. (A) HA-tagged proAct-Derrière was co-expressed in Xenopus oocytes along with either Flag-tagged proAct-Xnr2 or untagged proAct-Xnr2. When HA-tagged proAct-Derrière is expressed on its own and immunoprecipitated with HA, the mature ligand is seen as a broad range band between 22 and 28 kDa (lane 3). In the presence of Flag-tagged proAct-Xnr2, two distinct bands are apparent (lane 4); the removal of the Flag epitope tag from proAct-Xnr2 (a decrease in molecular weight of approximately 1 kDa) causes a corresponding mobility shift in the upper of the two bands (mature Xnr2 bands marked by asterisks). Note that co-expression of Derrière and Xnr2 also causes an overall decrease in the molecular weight of the mature Derrière ligand. (B) Xnr2 and BMP4 co-precipitate with Derrière. HA-tagged proAct-Derrière was expressed in oocytes either alone or in combination with Flag-tagged proAct-Derrière, proAct-Xnr2 or BMP4. Supernatants were analyzed by two-step immunoprecipitation; proteins were initially pulled down with HA coupled to protein A, released by boiling and immunoprecipitated a second time using Flag. Flag-tagged Derrière, Xnr2 and BMP4 are detected after the double immunoprecipitation (lanes 5-7), indicating that all of them associate with HA-tagged Derrière. All proteins are efficiently translated and secreted into the supernatant, as demonstrated by single immunoprecipitations (top two panels). (C) Wild-type Xnr2 co-precipitates with wild-type Derrière in oocyte lysates. Flag-tagged Derrière was expressed either alone or in combination with HA-tagged Derrière or HA-tagged Xnr2. Lysates were analyzed by HA and Flag immunoprecipitations. In both cases, unprocessed Xnr2 and Derrière were found to associate (lane 5). In addition, low levels of the mature Flag-tagged Derrière ligand co-precipitated with HA-tagged Xnr2 (arrowhead, bottom HA panel).