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Fig. 6. Pax3 can bind to but fails to transactivate the minimal limb
enhancer. Electrophoretic mobility shift assays, with in vitro translated
Pax3 (A), nuclear extract from limb buds and somites of mouse embryos
(B) and in vitro synthesized Meox2 (C). All shifts were
performed on the indicated wild-type oligonucleotide (wt). Specificity of
complex formation was assessed by competition with wild type or various mutant
oligonucleotides (mt1 to mt5) as outlined in A. Wild-type consensus motifs for
protein binding are highlighted by light gray and mutations by dark gray
boxes. Mutant mt5 has been chosen according to the Pax3-binding site proposed
in Bajard et al. (Bajard et al.,
2006). Protein complexes supershifted with specific antibodies are
indicated by arrows. (D) Transactivation of luciferase reporter
controlled by Myf5-XXI oligonucleotide sequence (WT, light columns) in
comparison to the mutant1 oligonucleotide (Mut, dark columns). 10T1/2 cells
were transfected with 200 ng of indicated luciferase reporter plasmids (wild
type or homeobox mutant), 200 ng expression vector encoding the indicated
transcription factors and 100 ng Renilla luciferase vector for transfection
control. The results are expressed as fold activation over transfection with
empty vector (pcDNA3.1) that had essentially no effect on either reporter.
Note the strong transcriptional activation that is dependent on the wild-type
homeobox motif by Pax3-VP16, while Pax3, Meox2 and both in combination are
only marginally active. Mean values of three independent transfections for
each construct are shown.
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