Fig. 2. Direct interaction of mCHL2 with BMPs, and inhibition of BMP4 binding to BMP receptor ectodomain by mCHL2. (A) FLAG-tagged CHL2 protein. Proteins in the peak eluate from the hydroxyapatite column chromatography were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and then silver stained (Sambrook et al., 1989). The mCHL2-FLAG band was excised and the NH₂-terminal amino acid sequence (vertical arrow in Fig. 1B) determined. (B) Immunoprecipitation/western blot analysis of mCHL2-FLAG individually mixed with BMP2 (a), BMP4 (b), BMP5 (c), BMP6 (d), BMP7 (e), GDF5 (f), activin A (g), TGFß1 (h), TGFß2 (i) or TGFß3 (j), followed by treatment with CHL2-COOH (lanes underlined). Immunocomplexes were detected using the corresponding antibodies (upper panels). Reactions only with mCHL2-FLAG, BMP, GDF, activin or TGFß were also performed as negative controls. Each blot was further developed with M2 to confirm the presence of precipitated mCHL2-FLAG (lower panels). The TGFß immunocomplexes (h-j) were separated into two sets; one was loaded on a non-reducing gel to visualize TGFß (upper panels), and the other on a reducing gel to detect CHL2 (lower panels). Lanes not underlined were directly loaded with the indicated amount (ng) of mCHL2-FLAG, BMP, GDF, activin or TGFß (for standards). (C) Inhibition of BMP4 binding to BMPR1B ectodomain by mCHL2-FLAG. The indicated amount of mCHL2-FLAG was first mixed with or without BMP4, and then BMPR1B-Fc or IgG-Fc was added. Protein complexes containing BMPR1B-Fc or IgG-Fc were selectively precipitated with protein A and subjected to western blot analysis (lanes underlined). Upper panel: bound BMP4 visualized with anti-BMP4 antibody. Middle panel: co-precipitation of mCHL2-FLAG checked with M2. Lower panel: precipitation of BMPR1B-Fc/IgG-Fc confirmed with anti-IgG-Fc antibody. For the standards, 0.04 µg of mCHL2-FLAG and 0.04 µg of BMP4 were loaded directly.