To determine if the GR directly binds the GRE #1 site, we utilized electrophoretic mobility shift assays (Figure 7). We observe a protein:DNA complex binding to the GRE #1 in both the 293-EcR and EcR-GR cells. There appears to be minimal modulation of the amount bound in the 293-EcR cells consistent with the expectation of binding by VgEcR-RXR with or without ligand present as is expected for the ecdysone-inducible system (Figure 7, lanes 12–15); however, we observe dexamethasone-dependent inhibition of binding in the EcR-GR cells (Figure 7, lanes 2–5 and 9–11). The amount of protein:DNA complex observed appears to be consistent with the results from the heterologous reporter assays (Figure 4). However, we were unable to demonstrate that the protein:DNA complex contains the GR as the addition of antibodies directed at the GR do not produce a supershift or substantially alter the relative levels of protein:DNA complex.
