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Fig. 2. Interactions of Gdf3 with activin receptors, Cripto and Lefty1. (A) Formation of a Gdf3 receptor complex. 293T cells were transfected with the indicated expression constructs, followed by crosslinking with the membrane-impermeable reagent DTSSP, immunoprecipitation of Myc-tagged ActRIIB from cell lysates and reversal of crosslinking; kinase-inactive activin receptor mutants were used in order to decrease receptor internalization. Western blots of immunoprecipitated and input proteins are shown, detected using the following antibodies: ${alpha}$ -Gdf3 (bf-Gdf3 proprotein, $~$ 49 kDa; mature bf-Gdf3, $~$ 17 kDa); ${alpha}$ -FLAG (f-Cripto, $~$ 21 kDa); ${alpha}$ -HA (ActRIB-HA, $~$ 54 kDa); ${alpha}$ -Myc (ActRIIB-6xMyc, $~$ 75 kDa). (B) Cripto interacts with Gdf3. Expression constructs were co-transfected into 293T cells, followed by crosslinking with DTSSP, immunoprecipitation of FLAG-tagged Cripto (f-Cripto) from cell lysates and reversal of crosslinking. Western blots are shown using the following antibodies: ${alpha}$ -V5 (bv-Gdf3 proprotein, $~$ 50 kDa) and ${alpha}$ -FLAG (f-Cripto, $~$ 21 kDa). (C) Gdf3 interacts with Lefty1 in conditioned media in the absence of crosslinking. The mature form of Lefty1 is preferentially immunoprecipitated; the expected positions of the Lefty1 proprotein (arrow) and a mature Lefty1 variant generated by use of an alternative prodomain cleavage site (asterisk) are indicated. Proteins were detected by western blotting using the following antibodies: ${alpha}$ -FLAG (bf-Gdf3 proprotein, $~$ 49 kDa; mature bf-Gdf3, $~$ 17 kDa) and ${alpha}$ -Lefty1 (Lefty1 proprotein, $~$ 40 kDa; mature Lefty1, $~$ 30 kDa).

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