FIG. 7.

Mechanism of BMP inhibition mediated by Rossy signaling in P19 cells. (A) Smad1 is phosphorylated in P19 cells expressing mRossy. GFP, green fluorescent protein; WB, Western blot. (B to E) Nuclear translocation of phosphorylated Smad1 following BMP4 stimulation is unaltered in P19 cells expressing an HA-tagged mRossy (C). (B) Unstimulated cells. (C) Cells stimulated with rhBMP4 protein for 30 min. (D and E) Expression of Rossy-HA in P19 cells stimulated with rhBMP4 (green signal) and of phospho-Smad1 (red signal). Arrows point to normal nuclear translocation of Smad1 in cells expressing Rossy (E). (F) Rossy expression inhibits the ability of activated Smad1/4 complex to bind the BRE element in an oligonucleotide pulldown assay (32). Notice the large decrease in immunoreactivity of Smad1 and Smad4 in the bound material. wt, wild type.