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Fig. 8. Barx2 binds to HBS elements within the Col2a intronic enhancer. (A) In
vitro translated Barx2 proteins (HD-BBRC) were tested for binding to the B1
and B2 probes in gel mobility-shift experiments. HD-BBRC protein formed
complexes with both the B1 and B2 probes that were completely blocked by
anti-Barx2 antibody. HD-BBRC did not bind to B1 and B2 probes in which ATTA
sequence was mutated (B1 HBS1 mut and B2 HBS2 mut). No complexes were formed
with control pcDNA3 extract. HD-BBRC formed complexes of similar intensity
with the B1 and B1 HMG5 mut probes. (B) Binding of the B1,B2 and S1 probes to
nuclear extracts from E12.5 embryonic limbs. The B1 probe formed stronger
complexes than either the B2 probe or the B1 HMG5 mut probe. Addition of Barx2
antibodies reduced binding to both probes, indicating that Barx2 is present in
the complex. Addition of Sox9 antibodies reduced binding to the probes that
contain HMG motifs (S1 and B1), suggesting that Sox9 is present in these
complexes. Sox9 antibody did not affect binding to the B2 probe. (C) Mutation
of HBS motifs in the B1 and B2 probes (B1 HBS1 mut and B2 HBS2 mut) abolished
their binding to limb nuclear extracts, whereas, mutation of the HMG motif in
the B1 probe (B1 HMG5 mut) reduced binding, suggesting that the HBS and HMG
sites can interact.
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