In order to determine if these binding site(s) were responsible for the VgEcR-RXR and GR mediated effects, the luciferase reporter assay was repeated with SEPP1 reporter constructs in which the two putative GREs or the RRE were mutated (Figure 5A). Despite the fact that GRE #2 was located within the −53 to +247 fragment that did not display any ponasterone A-induced luciferase activity in 293-EcR cells (Figure 2B), a mutant form of this binding site was tested. This GRE more closely matched the consensus sequence, with an inverted repeat of the GR binding site that could accommodate a GR homodimer, and therefore, could be involved in GR-mediated repression.
