Fig 2: FKBP51 overexpression increases RhoA activation. (a) 293T cells were transfected with the indicated expression vectors for 24 h and then subjected to the RhoA activation assay. Western blotting was completed after the pull‐down of activated forms of RhoA in cells expressing FKBP51 or DLC2. (b) Cells expressing FLAG‐FKBP51 or Halo‐DLC2 were visualized by anti‐Halo and anti‐FLAG antibodies. (c) Corresponding quantification from independent pull‐down experiments (normalization of the GTP‐bound forms to total RhoA) (left figure and column graph). Data in the column graph are represented as the mean ± SEM (n = 3; FLAG‐FKBP51 and FLAG‐mock for the RhoA pull‐down assay).

Fig 3: FKBP5, GR, and MR protein expression patterns in the mouse hippocampus(A) Coronal sections of C57BL/6J mice (n = 5) were stained for FKBP5 (FK506-binding protein 51), GR, and MR protein as well as DAPI (4′,6-diamidino-2-phenylindole). FKBP5 and MR exhibit similar expression patterns in the hippocampus, which is distinct from that of the GR. Scale bar, 250 μm.(B) Higher magnification images of the approximate CA1–CA2 boundary (white arrow) in the hippocampus. FKBP5 and MR expression is most prominent in hippocampal subregion CA2, whereas GR expression is strongly expressed in the CA1. Scale bar, 25 μm. See also Figure S1.

Fig 4: The Crh-specific overexpression of Fkbp5 in the PVN.A Crh-specific overexpression of Fkbp5 in the PVN was achieved by bilateral injections of a Cre-dependent Fkbp5 overexpression virus in the PVN of CRH-ires-CRE/Ai9 mice. B Validation of Crh-specific Fkbp5 mRNA overexpression in the PVN by ISH and RNAscope. Arrowheads pointing at viral Fkbp5 expressing Crh+ neurons (For mRNA quantification see Supplementary Fig. 9D). C Fkbp5CRH OE mice showed significantly increased adrenal weights (n = 10) and reduced thymus weights (n = 9) under non-stressed conditions compared to the controls (n = 10). D Corticosterone levels were mildly, but not significantly increased in Fkbp5CRH OE at 15, 30, and 60 min post-stress (Fkbp5CRH OE n = 10; Control n = 9). E Fkbp5CRH OE mice displayed a decrease in CRH neuronal mEPSC frequency accompanied by an increased amplitude (Fkbp5CRH OE nmouse = 4; nneuron = 29; Control nmouse = 4; nneuron = 25), which is reflected in two representative recording traces. Data are presented as mean ± SEM. All data were received from animals between 30 and 34 weeks of age and analyzed with the student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, T = 0.05 < p < 0.1, n.s. not significant.

Fig 5: GRs regulate FKBP5 expression under Dex-stimulated, but not under baseline, conditions in mouse primary hippocampal neuronsThe effects of altered GR levels on FKBP5 expression and on MR and GR binding to two Fkbp5-glucocorticoid response elements (GREs) were examined under baseline conditions and following dexamethasone (Dex) stimulation in primary hippocampal neurons, using biotinylated oligonucleotide immunoprecipitation (oligoIP).(A) Schematic summary of the experimental setup. After oligoIP, MR and GR binding to the Fkbp5-GRE oligonucleotide were quantified by western blotting using antibodies specific for the respective receptor. In addition, FKBP5, MR, or GR expression levels were quantified by western blotting from whole-cell extracts (WCEs).(B) Dex treatment increased FKBP5 levels in a concentration-dependent manner (F3,8 = 7.278, p < 0.05).(C and D) GR binding to the Fkbp5-GRE oligonucleotide was increased following Dex treatment (F3,8 = 8.9, p < 0.01), while MR binding was decreased (F3,8 = 10.35, p < 0.01).(E) Example blots of (B)–(D). Ctrl (control) 1: magnetic beads lacking conjugated streptavidin. Ctrl 2: cells treated with vehicle (Veh).(F) Transfection with a GR-expressing plasmid concentration dependently increased GR protein expression (F3,8 = 19.25, p < 0.001).(G–I) FKBP5 expression and MR and GR binding to the Fkbp5-GRE oligonucleotide are not altered following GR overexpression (OE) under baseline conditions.(J) Example blots of (F)–(I). Ctrl 1: magnetic beads lacking conjugated streptavidin. Ctrl 2: cells transfected with empty Ctrl vector.One-way analysis of variance (ANOVA) + Bonferroni post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean + SEM (n = mean derived from three independent experiments).