Figure 1. Interaction of FKBP51 with GRα and PPARγ Figure 1. Interaction of FKBP51 with GRα and PPARγ. GRα and PPARγ transfected COS-7 cells were adsorbed to protein G-Sepharose using the FiGR monoclonal antibody to GRα (I), PPARγ monoclonal antibody (I), or nonimmune IgG (NI), followed by Western blotting for GRα, PPARγ, Hsp90, and FKBP51 proteins. A representative of 3 independent experiments is shown. From: FKBP51 Reciprocally Regulates GRα and PPARγ Activation via the Akt-p38 Pathway Mol Endocrinol. 2014;28(8):1254-1264. doi:10.1210/me.2014-1023 Mol Endocrinol | Copyright © 2014 by the Endocrine Society

Figure 2. Reciprocal regulation of GRα and PPARγ activity by FKBP51 Figure 2. Reciprocal regulation of GRα and PPARγ activity by FKBP51. A, Receptor-less COS-7 cells were transfected with increasing concentrations of FKBP51 and assayed for FKBP51 expression by Western blotting. Hsp90 was used as a loading control. B, Cells of panel A were simultaneously transfected with expression constructs for GRα or PPARγ and analyzed for Dex-induced activity using a GRE-Luc reporter and Rosi-induced activity using PPRE-Luc (±SEM; n = 4–6). C, Western blot analysis of whole-cell extracts from WT and 51KO MEFs demonstrating a complete lack of FKBP51 in the 51KO cells. Hsp90 was used as a loading control. D, WT and 51KO cells were analyzed for GRα activity using GRE-Luc and PPARγ activity using PPRE-Luc in response to treatment with 1 μM Dex or 1 μM Rosi, respectively (±SEM; n = 3–6). All luciferase experiments used a pRL-CMV-Renilla reporter to normalize for transfection efficiency. Statistical differences are indicated as follows: *, P > .05; **, P > .01; ***, P > .001; the same parameters apply for # and ∧ symbols. *, WT vs WT; #, 51KO vs 51KO; ∧, WT vs 51KO. From: FKBP51 Reciprocally Regulates GRα and PPARγ Activation via the Akt-p38 Pathway Mol Endocrinol. 2014;28(8):1254-1264. doi:10.1210/me.2014-1023 Mol Endocrinol | Copyright © 2014 by the Endocrine Society

Figure 3. FKBP51 control of GRα and PPARγ phosphorylation Figure 3. FKBP51 control of GRα and PPARγ phosphorylation. A, 3T3–L1 preadipocytes were infected with lentivirus for stable expression of shRNAs against FKBP51 (FKBP1 knockdown [51KD]) or a scrambled control (Scr). Cells were grown to confluence, total protein was isolated, and expression of FKBP51 was analyzed by Western blotting. FKBP51 expression was normalized to Hsp90 (±SEM; n = 3). B, Whole-cell extracts of scramble and 51KD 3T3–L1 preadipocytes were analyzed by Western blotting with antibodies against total PPARγ or phosphoserine 112 (S112) of PPARγ. Phospho-PPARγ signals were normalized to total PPARγ (±SEM; n = 4). C, Whole-cell extracts of WT and 51KO MEFs treated with or without Dex (100 nM) for 1 hour were analyzed by Western blotting with antibodies specific to serines 212, 220, and 234 of mouse GRα. The FiGR antibody was used to detect total GRα. Phospho-GRα signals were normalized to total GRα (±SEM; n = 4). All quantitations were performed by infrared spectrophotometry. Statistical differences are indicated as follows: *, P > .05; **, P > .01; ***, P > .001; the same parameters apply for # and ∧ symbols. *, WT vs WT; #, 51KO vs 51KO; ∧, WT vs 51KO. From: FKBP51 Reciprocally Regulates GRα and PPARγ Activation via the Akt-p38 Pathway Mol Endocrinol. 2014;28(8):1254-1264. doi:10.1210/me.2014-1023 Mol Endocrinol | Copyright © 2014 by the Endocrine Society

Figure 4. FKBP51 regulates Akt phosphorylation at serine 473 and p38 MAPK activation. A, WT and 51KO MEFs treated with or without insulin (INS) for 30 minutes were analyzed by Western blotting with Akt antibodies specific to phosphoserine 473 or total Akt. B, WT and 51KO MEFs treated with or without insulin for 30 minutes were analyzed by Western blotting with a p38 antibody with dual specificity against phosphothreonine 180/phosphotyrosine 182 or an antibody against total p38 MAPK. C, WT and 51KO MEFs treated with or without Dex (100 nM) for 1 hour were analyzed by Western blotting with the p38 antibodies (see panel B). D, WT and 51KO MEFs treated with or without Dex for 1 hour were analyzed by Western blotting with Akt antibodies (see panel A). All quantitations were performed by infrared spectrophotometry and phospho-Akt levels were normalized to total Akt while phospho-p38 levels were normalized to total p38. Hsp90 was used as a loading control. All results represent means ± SEM (n = 4). Statistical differences are indicated as follows: *, P > .05; **, P > .01; ***, P > .001; the same parameters apply for # and ∧ symbols. *, WT vs WT; #, 51KO vs 51KO; ∧, WT vs 51KO. From: FKBP51 Reciprocally Regulates GRα and PPARγ Activation via the Akt-p38 Pathway Mol Endocrinol. 2014;28(8):1254-1264. doi:10.1210/me.2014-1023 Mol Endocrinol | Copyright © 2014 by the Endocrine Society

Figure 5. Insulin reciprocally regulates the intrinsic transcriptional activities of GRα and PPARγ. A, COS-7 cells were transiently transfected with GRα and GRE-Luc reporter in the absence or presence of insulin and Dex, as indicated. B, Similarly, COS-7 were transiently transfected with PPARγ and PPRE-Luc in the absence or presence of insulin and Rosi, as indicated. In each case, cells were transfected with pRL-CMV-Renilla to normalize for transfection efficiency. Final luciferase values were normalized to 1 for untreated cells. Results represent the means ± SEM (n = 6). *, control vs insulin; #, Dex or Rosi vs Dex or Rosi + insulin. Significant differences for all data are indicated as follows: *, P > .05; **, P > .01; the same parameters apply for #. From: FKBP51 Reciprocally Regulates GRα and PPARγ Activation via the Akt-p38 Pathway Mol Endocrinol. 2014;28(8):1254-1264. doi:10.1210/me.2014-1023 Mol Endocrinol | Copyright © 2014 by the Endocrine Society

Figure 6. p38 MAPK inhibition decreases GRα and increases PPARγ activity by reducing its phosphorylation. A, Analysis of GRα and PPARγ in PD169316 (PD)-treated COS-7 cells. COS-7 cells were transiently transfected with GRα and GRE-Luc reporter in the absence or presence of PD169316 and Dex, as indicated. Similarly, COS-7 were transiently transfected with PPARγ and PPRE-Luc in the absence or presence of PD169316 and Rosi, as indicated. In each case, luciferase activities were normalized to 1 for untreated cells. Results represent the means ±SEM (n = 6). *, Ctrl vs PD169316; #, Dex or Rosi vs Dex or Rosi + PD169316. B, Reversion of GRα and PPARγ activities in PD169316-treated 51KO MEFs. WT and 51KO cells were transiently transfected with GRα and GRE-Luc reporter or PPARγ and PPRE-Luc reporter, in the absence or presence of PD169316 and cognate ligand, as indicated. In each case, luciferase activities were normalized to 1 for untreated cells. Results represent the means ± SEM (n = 6). *, Ctrl vs Dex or Rosi; #, Dex or Rosi vs Dex or Rosi + PD169316. C, Inhibition of p38MAPK decreases PPARγ phosphorylation in 51KO cells. To increase PPARγ expression and activation, WT and 51KO cells were induced to differentiate into adipocytes, as described in companion paper (41). At day 5 postinduction, the extent of PPARγ phosphorylation was determined by Western blotting with antibodies against total PPARγ or phosphoserine 112 (S112) PPARγ in the absence or presence of PD169316, as indicated. Phospho-PPARγ signals were normalized to total PPARγ. Hsp90 was used as loading control. Results represent the means ± SEM (n = 3). *, WT control vs WT PD169316; #, 51KO control vs 51KO PD169316; ∧, WT control vs 51KO control. Significant differences for all data are indicated as follows: *, P > .05; **, P > .01; ***, P > .001; the same parameters apply for # and ∧ symbols. From: FKBP51 Reciprocally Regulates GRα and PPARγ Activation via the Akt-p38 Pathway Mol Endocrinol. 2014;28(8):1254-1264. doi:10.1210/me.2014-1023 Mol Endocrinol | Copyright © 2014 by the Endocrine Society

Figure 7. FKPB51 regulates steady-state intracellular localization of PPARγ to the cytoplasm. WT and 51KO MEFs were transfected with PPARγ-GFP in media containing charcoal-stripped serum to remove free fatty acids. DRAQ5 was used to label nuclear DNA. GFP and DRAQ5 were visualized 24 hours after transfection using a Leica DMIRE2 confocal microscope. Images shown are representative of 3 independent experiments where a minimum of 50 cells per condition were inspected. The fluorescence microscopy images were used with ImageJ to measure cellular localization of PPARγ-GFP. All results represent means ± SEM (n = 9). ****, P > .0001. From: FKBP51 Reciprocally Regulates GRα and PPARγ Activation via the Akt-p38 Pathway Mol Endocrinol. 2014;28(8):1254-1264. doi:10.1210/me.2014-1023 Mol Endocrinol | Copyright © 2014 by the Endocrine Society

Figure 8. Model for FKBP51 regulation of GRα and PPARγ Figure 8. Model for FKBP51 regulation of GRα and PPARγ. FKBP51 reciprocally modulates GRα and PPARγ by 2 distinct pathways. In the first, FKBP51 serves as a scaffold for recruitment of PHLPP phosphatase to Akt (15, 16), leading to Akt inactivation by dephosphorylation at serine 473. In turn, this reduces activation of p38 MAPK, leading to reduced stimulatory phosphorylation of GRα at serines 220 and 234, while simultaneously reducing the inhibitory phosphorylation of PPARγ at serine 112. In the second, FKBP51 serves to sequester both GRα and PPARγ to the cytoplasm, away from the active state of p38 MAPK in the nucleus. The net effect of both mechanisms is negative regulation of GRα, but positive regulation of PPARγ by FKBP51. Ask1, apoptosis signal-regulating kinase-1. From: FKBP51 Reciprocally Regulates GRα and PPARγ Activation via the Akt-p38 Pathway Mol Endocrinol. 2014;28(8):1254-1264. doi:10.1210/me.2014-1023 Mol Endocrinol | Copyright © 2014 by the Endocrine Society