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Fig. 1. Comparison of Gdf3 and Nodal signaling activities. (A) Schematic depiction of mouse Gdf3 and modified Gdf3 proteins; the sites of prodomain cleavage is indicated (arrows). (B) Gdf3 activity is Cripto and Foxh1 dependent. The indicated plasmids were co-transfected into 293T cells together with the A3-luc reporter. (Insets) Western blot detection of mature Nodal ( $~$ 12 kDa) and mature Gdf3 ( $~$ 17 kDa) proteins in conditioned media using ${alpha}$ -Nodal antibody or ${alpha}$ -Gdf3 antibody. (C) Gdf3 activity is facilitated by Cripto, but not Cryptic. 293T cells were co-transfected with expression vectors for Nodal or bf-Gdf3, Foxh1 and FLAG-epitope-tagged Cryptic, Cripto or Oep; control samples correspond to cells transfected with Nodal or bf-Gdf3 and Foxh1 without EGF-CFC proteins. (Inset) Western blot detection of EGF-CFC proteins in cell lysates using ${alpha}$ -FLAG antibody (FLAG-Cryptic, $~$ 22 and $~$ 20 kDa; FLAG-Cripto, $~$ 21 and $~$ 19 kDa; FLAG-Oep, $~$ 21 kDa). (D) Lefty1 inhibits Gdf3 activity. Cells were co-transfected with expression constructs for Nodal or bf-Gdf3, Cripto and Foxh1, in the presence or absence of Lefty1; controls correspond to these cells without Nodal or bf-Gdf3 transfection. (Inset) Western blot detection of mature Lefty1 protein ( $~$ 30 kDa) in conditioned media. In all panels, assays were performed in triplicate; error bars represent s.d.

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