Fig. 4.

Each cycle of GR reactivation is exclusively intranuclear and does not require transit through the cytoplasm. A, Treatment with okadaic acid (OA) after nuclear translocation was complete had no effect on the subsequent cyclical waves of GR activation measured by TransAM GRE binding of nuclear extract prepared from cells at each indicated time point. B, CHX inhibition of de novo protein synthesis had no effect on the subsequent intranuclear GR cyclical DNA association activity. C, All active transport through the nuclear pore was inhibited by transfection of the cells with the lectin WGA using the protein transfection reagent Chariot (Active Motif) after the initial pulse-directed GR translocation is complete. Subsequent intranuclear cyclical DNA association activity was unaffected by WGA transfection. The bacterial protein β-galactosidase (β-GAL) was used as a transfection control. D, Protein extracts from cells treated with CHX in part (B) were Western blotted to PVDF membrane and probed for the GC-inducible leucine zipper protein (GILZ) to verify de novo protein synthesis had been ablated. Clearly, this dose of CHX inhibited expression of GILZ protein during the time course of the experiment. E, Transfection of WGA before the first pulse of corticosterone inhibited nuclear translocation of GR and GRE binding in the TransAM assay. P, Pulse; W, wash.