Quantitative PCR results further qualified the induction of SEPP1 by VgEcR-RXR, and its repression by the GR (Figure 6A). In 293-EcR, gene induction of ~5 fold was observed following 24 hours of ponasterone A treatment, and dexamethasone treatment had no effect on this induction. Similar to the responses observed with the luciferase activity assays, the ability of ponasterone A to activate SEPP1 was attenuated in EcR-GR cells. SEPP1 expression was reduced by ~80% in these cells, even in the absence of dexamethasone treatment. Treatment with dexamethasone for 8 or 16 hours eliminated the ability of ponasterone A to induce gene expression, and led to additional repression of SEPP1 in a time dependent manner. In addition, immunochemical analysis of SelP from a Ni-NTA bead pull-down of the media from EcR-GR cells demonstrated a similar pattern of protein expression (Figure 6B).
