|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
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.
|
