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Fig. 3. The methyl-CpG binding function of kaiso is required to rescue the
xKaiso knockdown phenotype. The percentages of presented phenotypes
are indicated. (A) Embryos injected with 10 ng of CMO develop normally by
tadpole stage. (B) Over-expression of 750 pg wild-type human kaiso RNA does
not affect normal development of Xenopus embryos. (C) Injection of
750 pg of wild-type human kaiso RNA together with 5 ng KMO leads to complete
rescue of 26% of the embryos. (D) Apoptotic phenotype produced by injection of
5 ng KMO. (E) Co-injection of 750 pg of C522R human kaiso mutant RNA with 5 ng
KMO cannot rescue the phenotype of xKaiso depletion. (F) Location of
the kaiso mutant C522R amino acid substitution (red) in the third zinc finger,
leading to loss of the ability to bind methylated DNA. (G) Protein gel of
recombinant wild-type human kaiso and C522R (C>R) mutant proteins (arrow).
(H) Pull-down experiment showing p120ctn (arrow) binds both
wild-type human kaiso and C522R (C>R) mutant proteins in vitro, but not
human kaiso protein lacking the ZF domain (
ZF). (I) EMSA experiment
using recombinant C522R (C>R) mutant and wild-type human kaiso proteins
with methylated (lanes 1, 4), non-methylated Sm oligos (lanes 2, 5) and human
matrilysin (Hmat) oligo (lanes 3, 6) as probes. The kaiso-specific band shift
is arrowed. The C522R mutant shows no DNA-binding activity.
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