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Fig. 5. Identification of Pax3 binding sites on Pax3 promoters in vitro (A) Pax3, but not Zic1 alone, can trigger synthesis of the endogenous Pax3 transcript (primers in 3'UTR, not amplifying the inducible form) either in absence of the translation inhibitor cycloheximide (induction is 5-fold compared to uninjected animal caps) or in the presence of cycloheximide (induction is 31-fold, compared to cycloheximide-treated uninjected animal caps). Endogenous pax3 expression level in a stage 11 whole embryo is set at a relative value of 1 unit. After co-injection of Pax3 and Zic1, endogenous pax3 was activated as an immediate-early target as well (77-fold compared to uninjected animal caps; and, in presence of cycloheximide, 16-fold when compared to cycloheximide-treated uninjected animal caps). (B) Location of the ECR containing Pax3 putative binding site in the genomic sequence upstream of the Pax3 TSS. (C) Pax3 binds specifically to the motif identified in the pax3 promoter ECR: an electrophoretic mobility shift of the radiolabeled Pax3 binding site (Pax3BS) oligonucleotide probe is detected in the presence of Pax3-transfected cell extract, but not with the GFP-transfected cell extract. Intensity of the shift is reduced when the Pax3 binding site is mutated (mut. Pax3BS). In the presence of a specific anti-Pax3 antibody, we detect a supershift, indicating that the Pax3 protein is indeed responsible for the observed shift. Image published in: Plouhinec JL et al. (2014) Copyright © 2014. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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