Figure 5.

Failure to cleave at the S2 site leads to reduced levels of mature BMP-4. (A) One-cell embryos were injected with RNA encoding native or mutant myc-tagged proBMP-4, or with no RNA (ø) as indicated above each lane. Levels of precursor and ligand were examined by Western blot of embryonic extracts collected at the indicated Nieuwkoop and Faber (N/F) stages. Bands corresponding to uncleaved precursor and mature ligand are indicated to the right of the gel. The dark band in the center of the gel is a nonspecific signal that is present in uninjected embryos and serves as a loading control. (B) RNA encoding native or cleavage mutant forms of myc-tagged proBMP-4 were injected into a prospective ectodermal or mesodermal blastomere of 32-cell embryos and levels of mature BMP-4 examined by Western blot at stage 10 or 13. A representative blot with bands corresponding to mature ligand cleaved from the indicated precursors is shown. The level of expression of mature BMP-4 cleaved from native precursor in ectodermal cells (open bars) was arbitrarily set at 100% and expression of mature BMP-4 cleaved from native precursor in mesodermal cells, or from mutant precursors in either tissue was calculated relative to this value. The graph represents averaged values from three experiments.