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Fig. 5. Phosphorylated forms of BMP-dependent R-Smads colocalize with 52 bp
lacZ transgene expression in the limb and cardiac outflow tract, and
are recruited to the Msx2 BMP-responsive region in native chromatin.
Beads soaked in BMP4 were implanted on limbs of E11.5,
52bpMsx2-hsplacZ transgenic embryos. (A) Whole-mount lacZ
stain showing expression and BMP response in the anterior limb (arrow). (B-E)
Adjacent frozen sections along the dorsoventral axis of the limbs were either
stained for lacZ activity (B,C; blue against Nuclear Fast Red
counterstain), or immunostained with an antibody against the phosphorylated
forms of the BMP R-Smads 1, 5 and 8 (D,E; pink against DAPI counterstain).
(F-J) lacZ (G,I) and phospho-Smad (H,J) staining of adjacent
midsagittal sections through cardiac region of a 52bpMsx2-hsplacZ
embryo at E9.5 (F). A subset of phospho-Smad stained cells are positive for
lacZ activity. (K-M) ChIP assay showing association of phosphorylated
R-Smads with the Msx2 BMPRE in C14 limb mesenchymal cells. (K)
Autoradiogram of RNA derived from BMP2-treated C14 cells, probed for
Msx2. Upstream control and BMPRE primers (L) were used to interrogate
chromatin immunoprecitated with anti phospo-Smad1 antibody (M).
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