FIGURE 3.

Effects of FKBP51 overexpression on the nuclear transport of GRβ in NTM-5 and HeLa cells and on DEX-induced luciferase activity in HeLa cells. (A) Left: NTM-5 cells (~60% confluent) were transiently transfected with the empty vector pCMX, pCMV-FKBP51, or pCMX-hGRβ, as labeled. After posttransfection incubation for 24 hours, when the cell were completely confluent, cytosolic (100 µg) and nuclear (50 µg) fractions were prepared and subjected to coimmunoprecipitation with anti-FKBP51 antibody and SDS-PAGE, followed by Western immunoblot analysis with anti-GRβ antibody. Right: Western blot analysis for FKBP51 or GRβ in whole-cell lysates was used as a control for protein expression after transfection of pCMX, pCMV-FKBP51, or pCMX-hGRβ. (B) Left: HeLa cells (~60% confluent) were transiently transfected with pCMX, pCMV-FKBP51, or pCMX-hGRβ. After 24 hours of posttransfection incubation, cytosol and nuclear fractions were subjected to coimmunoprecipitation with anti-FKBP51 antibody and Western immunoblot analysis with anti-GRβ antibody. Right: Western immunoblot analysis for FKBP51 or GRβ in whole-cell lysates was used as a control for protein expression after transfection of pCMX, pCMV-FKBP51, or pCMX-hGRβ. (C) HeLa cells were cotransfected with 0.4 µg of glucocorticoid-responsive mercury luciferase reporter pGRE-Luc or 1.5 µg of empty vector pCMX, pCMV-FKBP51, or pCMX-hGRβ in 12-well culture slides. After a 24-hour incubation in complete medium, the cells were shifted to serum-free medium and treated with control vehicle or 100 nM DEX for an additional 24 hours. They were harvested, and luciferase activity was determined. Data are plotted as the x-fold change from each basal activation of vehicle treatment and represent the mean ± SEM of results in three independent experiments (*P < 0.05 pCMX+DEX versus pCMX+vehicle; pCMV-FKBP51+DEX versus pCMV-FKBP51+vehicle; pCMX-hGRβ+DEX versus pCMX-hGRβ+vehicle and **P < 0.05 pCMV-FKBP51+DEX versus pCMX+DEX; pCMX-hGRβ+DEX versus pCMX+DEX; one-way ANOVA).