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Fig. 6. The ISL sites in the Mef2c anterior heart field enhancer are
specifically bound by ISL1. Isl1 was transcribed and translated in
vitro and incubated with radiolabeled, double-stranded oligonucleotides
spanning the Mef2c ISL-p site (lanes 9-14), the Mef2c ISL-d
site (lanes 15-20) or a consensus ISL1 binding site from the rat Insulin
I gene as a control (lanes 1-8). ISL1 efficiently bound to all three ISL
sites (lanes 2, 10 and 16). Binding of ISL1 to the ISL-p site was specifically
competed by excess unlabeled Mef2c ISL-p (I-p) (lane 11) and by
excess unlabeled Insulin I ISL1 site (In) (lane 13) but not by a
100-fold excess of mutant ISL-p site (mI-p) (lane 12) or by a 100-fold excess
of a mutant Insulin I site (mIn) (lane 14). Likewise, the binding of
ISL1 to the ISL-d site was specifically competed by excess unlabeled ISL-d
(I-d) (lane 17) and by excess unlabeled Insulin I site (lane 19) but
not by a 100-fold excess of either mutant ISL-d (mI-d) (lane 18) or mIn
control (lane 20). The Mef2c ISL-p and ISL-d sites also efficiently
competed for ISL1 binding to the control ISL1 site from the Insulin I
promoter (lanes 5 and 7), but mutant versions of the ISL-p and ISL-d sites
were unable to compete for binding to the Insulin I site (lanes 6 and
8). In samples where in vitro translated ISL1 protein was not included
(denoted by a minus sign in lanes 1, 9 and 15), an equal amount of
unprogrammed reticulocyte lysate was included.
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