For immunoprecipitation assays, HEK293T cells were transfected with the indicated plasmids. Cells were harvested 48 hours after transfection and lysed with TNE lysis buffer [10 mM tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, and 0.5% Nonidet P-40] containing a protease inhibitor cocktail. Immunoprecipitation assays were performed on lysates and collected CM as previously described (39).

For immunoblotting, affinity-purified anti-Flag (F2555, Sigma), anti-HA (CW0092A, CW), anti-Myc (M047-3, MBL), anti–phospho-Smad1/5/8 (9511, Cell Signaling Technology), anti-Smad1/5/8 (ab72504, abcam), anti–phospho-Smad2/3 (3101, Cell Signaling Technology), anti-Smad2/3 (3102, Cell Signaling Technology), anti-Chd (GTX128209, GeneTex), and anti–β-actin (SC1615, Santa Cruz Biotechnology) antibodies were used.

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