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. 2013 May 17;27(7):1020–1035. doi: 10.1210/me.2012-1023

Figure 5.

Figure 5.

GRs containing guinea pig H1-H3 loop mutations are more sensitive than wild-type (WT) receptor to repression by FKBP51 without an increased association with the cochaperone. A, MEF FKBP51-KO cells were transfected with a reporter plasmid pGRE.luc and a receptor expression plasmid for either rGR or mutated rGR. Cells were treated with increasing concentrations of cortisol or ethanol vehicle for 20 hours before being harvested. Luciferase values were standardized against protein and plotted as a percentage of the maximum observed response to hormone. Each graph represents the mean ± SEM of 3 independent experiments, *, P < .05 for comparison of transcriptional activity between wild-type and mutant rGR. B, MEF FKBP51-KO cells were transfected as above with the addition of pcDNA3.1/hFKBP51 expression plasmid or empty vector control. Cells were treated with 50 nM cortisol or ethanol vehicle for 20 hours before being harvested. Luciferase values were standardized against protein and plotted as the fold change of receptor activity in response to hormone treatment. *, P ≤ .002 for comparison of fold change in transcriptional activity for wild-type versus mutant rGR in cells overexpressing FKBP51. C, HEK293 cells were transfected with a reporter plasmid pGRE.luc, a receptor expression plasmid for either rGR or mutated rGR and pRK5MCS/FKBP51-FLAG expression plasmid or empty vector control. Cells were treated with 50 nM cortisol or ethanol vehicle for 20 hours before being harvested. *, P ≤ .035; **, P ≤ .001; for comparison of fold change in transcriptional activity for wild-type or mutant rGR in cells containing the empty vector control versus overexpression of FKBP51-FLAG. D, Representative blot of 3 independent experiments where HEK293 cells were transfected with plasmids expressing HA-rGR (wild-type or mutant [IHS/TS]) and either FKBP51-FLAG or empty vector control. After 60 hours incubation, the cells were lysed and protein extracts immunoprecipitated (IP) with anti-HA agarose conjugate. The comparative levels of FKBP51-FLAG that coimmunoprecipitated with wild-type and mutant HA-rGR, together with the levels of HA-rGR bound to the beads, were assessed by anti-FLAG and anti-HA Western blot analysis, respectively (upper panel). The relative levels of input protein in the lysates are shown in the lower panel.