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Fig. 2. Tbx6 binds to P2PSME in the PSM and tailbud. (A,B)
Characterization of the anti-Tbx6 antibody produced in this study. (A)
Whole-mount immunohistochemistry demonstrating the localization of Tbx6
protein in the mouse PSM and tailbud. (B) Western blot analysis showing that
the anti-Tbx6 antibody detected a protein of expected molecular weight (58
kDa) in the PSM and tailbud (p) but not in formed somites (s). The asterisk
indicates non-specific binding. (C) Double staining of Mesp2
mRNA (purple) and Tbx6 protein (green) demonstrating the coexistence of both
signals in the anterior-most part of the Tbx6-positive region (white in merged
image). (D) Design of an in vivo technique for detecting Tbx6 binding
to the P2PSME by ChIP. Arrows represent primers for the ChIP assay for the
Mesp2 and Dll1 genes. Dll1 is known to be
downstream of Tbx6 and was therefore used as a positive control. Gray and
black boxes represent the P2PSME and Dll1 mesoderm (msd) enhancers,
respectively. White ovals indicate Tbx6 binding sites. P2Em and P2Ewt, mutated
and wild-type P2PSME regions, respectively; NC, unrelated sequence as negative
control. (E) Tbx6 associates with P2PSME in the anterior and posterior
PSM. (F) The association of Tbx6 with mutated P2PSME as detected by
ChIP assay. Mutated and wild-type P2PSME regions were differentially detected
by PCR with different sets of primers in the tails of E10.5 embryos obtained
from the crossing of P2EmB1D/+ and ICR mice.
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