Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 1. Effect of FKBP51 knockdown on NF-B activation and apoptosis sensitivity in A375 melanoma cells. (A, upper) IB assay of IB phosphory- lation levels in KA samples. IKK complexes were immunoprecipitated from melanoma cells stably knocked down with three different FKBP51 shRNAs (1, 2 and 3) or with a control shRNA (Ctrl) and stimulated with TNF for 15 and 30 min. GST-IB served as substrate (top panel) to measure IKK activity. IB assay also monitored the IKK levels in immunoprecipitated-protein. Whole lysates showed the efficacy of FKBP51 knock down. HSP60 was used as a loading control. The reduced level of GST-p-IB in condition of FKBP51 knock down is consistent with a reduced phosphorylating capacity of immunoprecipitated IKK. (A, lower) EMSA of nuclear extracts of FKBP51-knocked down A375 cells, stimulated with TNF for 30 and 60’. The band was generated by NF-B binding to a 32P-radiolabeled probe. The bands indicated by the arrow are reduced in FKBP51 knock down cells. Nuclear extract was normalized using lamin B as a loading control. The expression of FKBP51 and -actin in total lysates is also shown. (B) Assay of TNF-induced changes in expression levels of IB protein (IB, upper) and mRNA (qPCR, lower). (C and D) Effect of FKBP51 knock down on TNF-induced apop- tosis. (C) Expression levels (protein and mRNA) of pro-apoptotic Bax. (D) Representative flow cytometric histograms of annex-V/PI staining. Data are representative of three independent experiments.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Knockdown, Activation Assay, Immunoprecipitation, Stable Transfection, Control, shRNA, Activity Assay, Generated, Binding Assay, Expressing, Staining

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 2. FKBP51 interacts with all IKK complex subunits and supports IKK assembly. (A) Immunoassay of HEK293 cells transfected with several com- binations of expression vectors, namely HA-IKK, -IKK, -IKK, -TAK1 and Flag-FKBP51. Anti-Flag immunoprecipitated proteins were IB assayed with anti-HA and anti-Flag (upper). IB analysis of total lysates is also shown (lower). (B and C) FKBP51 knock down impairs the IKK/IKK/IKK interaction. (B) Immunoassay of HEK293 cells, silenced or not for FKBP51, and transfected with the expression vectors for IKK, HA-IKK and Flag- FKBP51. IKK was immunoprecipitated from cell lysates; IP was analyzed by IB with anti-HA. (C) Endogenous IKK was immunoprecipitated from whole lysates of stably knocked down A375 melanoma cells (1,2) and the relative negetive control (C). Immunoprecipitated proteins were then assayed by IB. Data are representative of three independent experiments.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Transfection, Expressing, Immunoprecipitation, Knockdown, Stable Transfection, Control

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 3. FKBP51 isomerase activity affects the enzymatic function of IKK kinase complex. (A) IB of IB phosphorylation levels of samples assayed in KA. IKK complexes were immunoprecipitated from WT and FKBP51-knocked down A375 cells, stimulated with TNF for 30 and 45 min, in the presence or not of FK506. IKK activation was measured using GST-IB as substrate. IB also monitored the expression of IKK in immunoprecipitated protein and FKBP51 in total lysates. HSP60 served as loading control for total lysates. Data are representative of three independent experiments. (B) Immunoassay of HEK293 cells transfected with the HA-IKK and Flag-FKBP51 expression vectors, in the presence or not of FK506. Data are representative of two independent experiments. (C) Study of IB degradation induced by TNF in A375 cells pre-incubated for 1 h in the absence or the presence of FK506, rapamycin and CSA, each compound at the concentration of 0.2 g/ml. Data are representative of two independent experiments. (D) IB degradation induced by TNF- in A375 cells pre-incubated for 1h in the absence or the presence of Rapamycin, CSA and the specific FKBP51 inhibitors SaFit1 and 2, each at the concentration of 0.2 g/ml. Data are representative of two independent experiments. Both specific compounds inhibited TNF--induced IB degradation. (E) Measure by qPCR of CCND1 and FKBP1A levels showed that both SaFit1 and 2 significantly reduced TNF--induced of CCND1 levels of melanoma, in accordance with the NF-B promoting ability of FKBP51. Modulation of cyclin D1 expression level was confirmed by immunoblot. Neither FKBP1A mRNA, nor FKBP12 protein, appeared to be modulated.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Activity Assay, Phospho-proteomics, Immunoprecipitation, Activation Assay, Expressing, Control, Transfection, Incubation, Concentration Assay, Western Blot

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 4. FKBP51 regulates interaction between TRAF2 and IKK. (A) Reduced TRAF2 levels in FKBP51 knocked down melanoma cells. QPCR analysis of the TRAF2 and FKBP51 mRNA levels in FKBP51-knocked down A375 cells. Data are representative of three independent experiments. (B) FKBP51 interacts with TRAF2. Immunoassay of HEK293 cells transfected with the HA-TRAF2 and Flag-FKBP51 expression vectors. Data are representative of three independent experiments. (C) FKBP51 silencing prevents TRAF2 interaction with IKK subunits. Immunoassay of whole cell extracts and im- munoprecipitated proteins obtained by HEK293, silenced or not for FKBP51, and transfected with various combinations of HA-IKK, -IKK, -IKK, -TAK1, -TRAF2 and Flag-FKBP51 expression vectors. Data are representative of four independent experiments. (D) FKBP51, IKK subunits and TRAF2 interact each other. Endogenous IKK and FKBP51 were immunoprecipitated from whole lysates of A375 melanoma cells. Immunoprecipitated protein was then assayed in IB. Data are representative of two independent experiments.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Transfection, Expressing, Immunoprecipitation

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 5. FKBP51 domains involved in binding to TRAF2 and IKK subunits. (A) Mutation of TPR domain impairs FKBP51/TRAF2 interaction. Immunoassay of HEK293 cells transfected with HA-TRAF2, Flag-FKBP51-mutTPR (carrying point mutation of TPR), FKBP51-mutPPIase (carrying point mutation of PPIase), Myc-Flag-FKBP51s (a truncated FKBP51 isoform lacking of TPR domains). Lysates were subjected to immunoprecipitation with anti-Flag and IB analysis was performed with anti-TRAF2 and anti-Flag (upper). IB of total lysates is also shown (lower). (B) TPR domain of FKBP51 promoted the association between the IKK subunits. Immunoassay of HEK293 cells transfected with the HA-TRAF2, -IKK, -IKK and Flag- FKBP51-mutants expression vectors. Lysates were subjected to immunoprecipitation with anti-Flag, or anti-IKK and analyzed by IB with anti-IKK/ and anti-TRAF2. IB of whole lysates is also shown. (C) Both TPR and PPIase domains of FKBP51 are involved in the IKK/FKBP51 interaction. Immunoassay of HEK293 cells transfected with HA-IKK and differents Flag-FKBP51 mutants. Lysates were subjected to immunoprecipitation with anti-IKK and analyzed by IB with anti-Flag. IB analysis of whole lysates is also shown. Data are representative of three independent experiments.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Binding Assay, Mutagenesis, Transfection, Immunoprecipitation, Expressing

Journal: Nucleic acids research

Article Title: FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma.

doi: 10.1093/nar/gkv615

Figure Lengend Snippet: Figure 6. Proposed mechanism for the interaction of FKBP51 with NF-B signaling proteins. (A) TNF-binding to its receptor determines formation of RIP-induced K-63 ubiquitin chain. (B) TRAF2, promotes elongation of this non degradative ubiquitin chain and recruits TAK1 kinase complex. (C) TRAF2 interacts with TPR domain of FKBP51. IKK interacts with both FK and TPR domains of FKBP51. IKK and TRAF2 are also connected through K63 ubiquitin chain. (D) IKK and interact each others and with IKK and TRAF2 through the TPR domain.

Article Snippet: Antibodies used for co-IP: anti-FKBP51 antibodies were from Novus Biologicals (rabbit polyclonal, NB100-68240) and from Abnova (mouse polyclonal H00002289-B01P, Taipei, Taiwan); anti-IKK antibodies were from Santa Cruz (rabbit polyclonal anti-IKK , H-744; rabbit polyclonal anti-IKK / , H-470).

Techniques: Binding Assay, Ubiquitin Proteomics

Journal: Molecular Cancer Therapeutics

Article Title: Cell Intrinsic Role of COX-2 in Pancreatic Cancer Development

doi: 10.1158/1535-7163.mct-12-0342

Figure Lengend Snippet: Figure 7. Decreased FKBP5 negative feedback leads to enhanced AKT activation in Pdx1-Creþ;K-rasG12D/þ;Ptenlox/þ; Cox-2lox/lox mice. A, FKBP5 mRNA expression in PDACs of Pdx1-Creþ; K-rasG12D/þ;Ptenlox/þ;Cox-2lox/lox

Article Snippet: The following primary antibodies were used: phospho-AKT(Ser473) (Cell Signaling; 1:50), Cytokeratin 19 (ab15463, Abcam; 1:100), COX-2 (SP21; Thermo Scientific, ready-to-use),GRP78(11587-1AP,ProteinTechGroup; 1:50), and FKBP5 (14155-1-AP, ProteinTech Group, 1:50).

Techniques: Activation Assay, Expressing

Journal: PLoS Medicine

Article Title: Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans

doi: 10.1371/journal.pmed.1001755

Figure Lengend Snippet: (A and B) Two batches of 51KO and wild-type mice were treated with PAR (10 mg/kg) or vehicle (A), or with AMI (10 mg/kg) or vehicle (B) and subjected to the FST ( n = 7–9 per group). Graphs show the times of immobility and struggling. (C–G) 51KO and wild-type mice were treated with PAR (10 mg/kg) or vehicle and sacrificed 45 min later. The levels of the indicated proteins were determined in extracts of the hippocampus and the prefrontal cortex from 9–11 animals per group. (H–K) Similar experiments were performed with AMI (10 mg/kg) instead of PAR. (L) Deletion of FKBP51 does not change blood-brain barrier function. 51KO and wild-type mice were treated with AMI (10 mg/kg, n = 5 for both wild-type and 51KO mice) or PAR (10 mg/kg, n = 4 for wild-type mice, n = 3 for 51KO mice) and sacrificed 45 min later. Levels of PAR, AMI, and the active AMI metabolite nortriptyline (NOR) were determined in blood and brain. Graphs display the ratio of the concentration in the brain (nanograms of drug per gram of brain tissue) and the concentrations in the plasma (nanograms of drug per milliliter of plasma). * p <0.05; ** p <0.01; *** p <0.001. Statistical parameters in .

Article Snippet: The following primary antibodies were used: Beclin1 (1∶1,000, Cell Signaling, #3495), pBeclin1 (S234 and S295, both 1∶1,000, Phosphosolutions, #p117-234 and #p117-295), Atg12 (1∶1,000, Cell Signaling, #2010), LC3B-II/I (1∶1,000, Cell Signaling, #2775), FLAG (1∶7,000, Rockland, 600-401-383), PI3K Class III (Vps34, 1∶1,000, Cell Signaling, #4263), FKBP51 (1∶1,000, Bethyl, A301-430A), Akt (1∶1,000, Cell Signaling, #4691), pAkt (Ser473 and T308, both 1∶1,000, Cell Signaling, #4058 and #9275), Actin (1∶5,000, Santa Cruz Biotechnology, sc-1616).

Techniques: Concentration Assay, Clinical Proteomics

Journal: PLoS Medicine

Article Title: Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans

doi: 10.1371/journal.pmed.1001755

Figure Lengend Snippet: (A) FKBP51 interacts with Beclin1, Akt, and PHLPP. HEK cells were transfected with vector control (lanes 1 and 3) or a FLAG-tagged FKBP51-expressing plasmid (lanes 2 and 4) and lysed 72 h later. After immunoprecipitation (IP) of protein complexes using a FLAG antibody, input (lanes 1 and 2) and (co)precipitated (lanes 3 and 4) proteins were visualized by Western blotting. (B) Change of total Beclin1, pAkt S473 , and pAkt T308 upon expression of FKBP51. Representative Western blots on the right. FKBP51 was detected by an antibody directed against its FLAG tag. (C) FKBP51 interacts preferentially with dephosphorylated Akt and promotes Akt dephosphorylation. Graphs display quantification of pBeclin1 (S234 and S295) and Beclin1-Akt/pAkt S473 interaction after Beclin1 immunoprecipitation from HEK cells transfected with FKBP51/52 (details in ). (D) Comparison of the endogenous protein levels of effects of Beclin1, LC3B-II/I, Atg12, and pAkt S473 levels in wild-type MEFs, 51KO MEFs, and 51KO MEFs transfected with FKBP51 or vector control. Graphs show the relative expression, with the levels of wild-type cells transfected with vector control set to 1 (dashed line). Representative Western blots on the right. (E) Quantification of Beclin1-Akt interaction after Beclin1 immunoprecipitation in brain extracts from wild-type and 51KO mice. Graph represents the results from five independent experiments. Representative Western blots on the right. (F) FKBP51 enhances autophagic flux. Cortical rat astrocytes were transfected with FKBP51 or vector and treated with bafilomycin A 1 (BafA1) as indicated, and the levels of LCB3-II/I were determined. (G) Cellular effects of FKBP51 on autophagic markers require Akt1 and/or Akt2. The protein levels of Beclin1 and LC3B-II/I were evaluated in wild-type MEFs, Akt1/2KO MEFs, and Akt1/2KO MEFs transfected with Akt1- and Akt2-expressing plasmids. Graphs display the relative expression of three different experiments; expression in wild-type vector-transfected MEFs was set to 1 (dashed line). Representative Western blots on the right. * p <0.05; ** p <0.01; *** p <0.001. See for statistical details. ect., ectopic; genot., genotype; KO, 51KO; WT, wild-type.

Article Snippet: The following primary antibodies were used: Beclin1 (1∶1,000, Cell Signaling, #3495), pBeclin1 (S234 and S295, both 1∶1,000, Phosphosolutions, #p117-234 and #p117-295), Atg12 (1∶1,000, Cell Signaling, #2010), LC3B-II/I (1∶1,000, Cell Signaling, #2775), FLAG (1∶7,000, Rockland, 600-401-383), PI3K Class III (Vps34, 1∶1,000, Cell Signaling, #4263), FKBP51 (1∶1,000, Bethyl, A301-430A), Akt (1∶1,000, Cell Signaling, #4691), pAkt (Ser473 and T308, both 1∶1,000, Cell Signaling, #4058 and #9275), Actin (1∶5,000, Santa Cruz Biotechnology, sc-1616).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Immunoprecipitation, Western Blot, FLAG-tag, De-Phosphorylation Assay, Comparison

Journal: PLoS Medicine

Article Title: Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans

doi: 10.1371/journal.pmed.1001755

Figure Lengend Snippet: (A) The interaction of FKBP51 with Beclin1 and Akt in HEK cells in the presence or absence of PAR (10 µM, 72 h) was analyzed by CoIP. (B) Primary rat cortical astrocytes were transfected with vector control or FKBP51 and treated with PAR (10 µM) for 72 h, and the pAkt S473 /Akt ratio was determined. (C and D) Wild-type MEFs, 51KO MEFs, and 51KO MEFs transfected with FKBP51 or vector control were treated with PAR (10 µM) for 72 h; pAkt S473 /Akt (C) and LC3B-II/I (D) ratios were determined. Protein levels in untreated wild-type MEFs were set to 1 (dashed line). (E) Primary astrocytes were cotransfected with GFP-LC3B and vector control or FKBP51, and treated with PAR (10 µM) for 72 h. The number of GFP-LC3B-positive puncta/cell was determined in 15–25 randomly selected cells for each condition. (F) Representative fluorescence images of (E). (G–I) Primary astrocytes were transfected with vector control or FKBP51, and treated as in (B); Beclin1, LC3B-II/I, and Atg12 levels were determined. Protein levels of untreated vector-transfected cells were set to 1 (dashed line). (J) Antidepressants enhance autophagic flux. Rat cortical astrocytes were incubated with the drugs as indicated for 2 h, and the levels of LC3B-I, LC3B-II, and Actin were determined in protein extracts by Western blotting. The graph displays the median ratio of LC3B-II/I from three independent experiments. * p <0.05; ** p <0.01; *** p <0.001. Statistical parameters in . BafA1, bafilomycin A 1 ; ect., ectopic; genot., genotype; IP, immunoprecipitation; KO, 51KO; RAP, rapamycin; WT, wild-type.

Article Snippet: The following primary antibodies were used: Beclin1 (1∶1,000, Cell Signaling, #3495), pBeclin1 (S234 and S295, both 1∶1,000, Phosphosolutions, #p117-234 and #p117-295), Atg12 (1∶1,000, Cell Signaling, #2010), LC3B-II/I (1∶1,000, Cell Signaling, #2775), FLAG (1∶7,000, Rockland, 600-401-383), PI3K Class III (Vps34, 1∶1,000, Cell Signaling, #4263), FKBP51 (1∶1,000, Bethyl, A301-430A), Akt (1∶1,000, Cell Signaling, #4691), pAkt (Ser473 and T308, both 1∶1,000, Cell Signaling, #4058 and #9275), Actin (1∶5,000, Santa Cruz Biotechnology, sc-1616).

Techniques: Transfection, Plasmid Preparation, Control, Fluorescence, Incubation, Western Blot, Immunoprecipitation

Journal: PLoS Medicine

Article Title: Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans

doi: 10.1371/journal.pmed.1001755

Figure Lengend Snippet: Wild-type and 51KO mice were subjected to CSDS and subsequent chronic PAR treatment resulting in eight experimental groups ( n = 8–12 per group) that were analyzed for behavior and autophagy marker protein levels. When the three-way ANOVA indicated an interaction effect ( p <0.1), genotype-dependent stress and treatment effects were isolated by normalizing the data to either unstressed controls or vehicle treatment. (A) General locomotion in the open-field arena was independent of genotype, condition, or treatment of the mice. (B–D) Deletion of FKBP51 abolished the reaction to CSDS and to chronic PAR treatment in the social avoidance test. (E–H) Deletion of FKBP51 significantly reduced the effect of chronic PAR treatment in the FST. (I–R) Deletion of FKBP51 abolished the effects of chronic PAR treatment on the autophagy pathway markers Beclin1, Atg12, pAkt, and LC3B-II/I. Protein parameters were analyzed in hippocampal brain extracts. Asterisks indicate significant result for planned contrast test for main genotype effect (#), main treatment effect ($), or main condition effect (+): * p <0.05; ** p <0.01. a.u., arbitrary units; SI zone, social interaction zone; WT, wild type.

Article Snippet: The following primary antibodies were used: Beclin1 (1∶1,000, Cell Signaling, #3495), pBeclin1 (S234 and S295, both 1∶1,000, Phosphosolutions, #p117-234 and #p117-295), Atg12 (1∶1,000, Cell Signaling, #2010), LC3B-II/I (1∶1,000, Cell Signaling, #2775), FLAG (1∶7,000, Rockland, 600-401-383), PI3K Class III (Vps34, 1∶1,000, Cell Signaling, #4263), FKBP51 (1∶1,000, Bethyl, A301-430A), Akt (1∶1,000, Cell Signaling, #4691), pAkt (Ser473 and T308, both 1∶1,000, Cell Signaling, #4058 and #9275), Actin (1∶5,000, Santa Cruz Biotechnology, sc-1616).

Techniques: Marker, Isolation

Journal: PLoS Medicine

Article Title: Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans

doi: 10.1371/journal.pmed.1001755

Figure Lengend Snippet: Protein levels of FKBP51 and Beclin1 (A), pAkt S473 /Akt (B), LC3B-II/I (C), and Atg12 (D) in PBMCs from healthy men ( n = 21). Each dot represents the levels of FKBP51 and the respective protein in the PBMCs from one individual. Average expression levels were set to 1.

Article Snippet: The following primary antibodies were used: Beclin1 (1∶1,000, Cell Signaling, #3495), pBeclin1 (S234 and S295, both 1∶1,000, Phosphosolutions, #p117-234 and #p117-295), Atg12 (1∶1,000, Cell Signaling, #2010), LC3B-II/I (1∶1,000, Cell Signaling, #2775), FLAG (1∶7,000, Rockland, 600-401-383), PI3K Class III (Vps34, 1∶1,000, Cell Signaling, #4263), FKBP51 (1∶1,000, Bethyl, A301-430A), Akt (1∶1,000, Cell Signaling, #4691), pAkt (Ser473 and T308, both 1∶1,000, Cell Signaling, #4058 and #9275), Actin (1∶5,000, Santa Cruz Biotechnology, sc-1616).

Techniques: Expressing

Journal: PLoS Medicine

Article Title: Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans

doi: 10.1371/journal.pmed.1001755

Figure Lengend Snippet: (A and B) PBMCs were collected from healthy subjects ( n = 14) before and 6 h after the intake of 1.5 mg of DEX, and proteins were analyzed. Each dot represents the DEX-induced change in FKBP51 and the expression change of the respective protein (Beclin 1 [A] and pAkt S473 [B]) in the PBMCs of one individual. (C–F) Levels of FKBP51 and Beclin1 (C), pAkt S473 (D), LC3B-II/I (E), and Atg12 (F) in PBMCs from healthy men ( n = 21) cultivated ex vivo and treated with PAR (0.365 µM, 48 h). Plots depict protein changes upon treatment with antidepressant compared to vehicle-treated cells in correlation to FKBP51. Each dot represents the level of FKBP51 and the expression change of the respective protein in the PBMCs from one individual.

Article Snippet: The following primary antibodies were used: Beclin1 (1∶1,000, Cell Signaling, #3495), pBeclin1 (S234 and S295, both 1∶1,000, Phosphosolutions, #p117-234 and #p117-295), Atg12 (1∶1,000, Cell Signaling, #2010), LC3B-II/I (1∶1,000, Cell Signaling, #2775), FLAG (1∶7,000, Rockland, 600-401-383), PI3K Class III (Vps34, 1∶1,000, Cell Signaling, #4263), FKBP51 (1∶1,000, Bethyl, A301-430A), Akt (1∶1,000, Cell Signaling, #4691), pAkt (Ser473 and T308, both 1∶1,000, Cell Signaling, #4058 and #9275), Actin (1∶5,000, Santa Cruz Biotechnology, sc-1616).

Techniques: Expressing, Ex Vivo

Journal: PLoS Medicine

Article Title: Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans

doi: 10.1371/journal.pmed.1001755

Figure Lengend Snippet: Beclin1, FKBP51, LC3B-II/I, and pAkt S473 /Akt levels were determined in PBMCs from inpatients with depression at admission ( n = 51). (A–C) Expression values of Beclin1 (A), pAkt S473 /Akt (B), and FKPP51 (C) are correlated with the response to antidepressant (AD) treatment expressed as percent change in total score on the 21-item HDRS between admission and after 6 wk of treatment. (D–F) Expression values of the autophagy pathway markers Beclin1 (D), pAkt S473 /Akt (E), and LC3B-II/I (F) are correlated with the expression levels of FKBP51.

Article Snippet: The following primary antibodies were used: Beclin1 (1∶1,000, Cell Signaling, #3495), pBeclin1 (S234 and S295, both 1∶1,000, Phosphosolutions, #p117-234 and #p117-295), Atg12 (1∶1,000, Cell Signaling, #2010), LC3B-II/I (1∶1,000, Cell Signaling, #2775), FLAG (1∶7,000, Rockland, 600-401-383), PI3K Class III (Vps34, 1∶1,000, Cell Signaling, #4263), FKBP51 (1∶1,000, Bethyl, A301-430A), Akt (1∶1,000, Cell Signaling, #4691), pAkt (Ser473 and T308, both 1∶1,000, Cell Signaling, #4058 and #9275), Actin (1∶5,000, Santa Cruz Biotechnology, sc-1616).

Techniques: Expressing

Journal: PLoS Medicine

Article Title: Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans

doi: 10.1371/journal.pmed.1001755

Figure Lengend Snippet: FKBP51 interacts with PHLPP, Akt, and Beclin1. Since PHLPP dephosphorylates Akt (S473 in Akt1), inactive Akt is recruited to Beclin1. This results in lower phosphorylation of Beclin1 and, thus, the induction of autophagic pathways . Autophagic pathways, and thereby also FKBP51, are linked to cell homeostasis and to synaptic (syn.) function as a physiological correlate of behavior . Antidepressants act on the same pathways in an FKBP51-dependent manner and change FKBP51 protein interactions; this could form the basis for the FKBP51 dependency of antidepressant effects in cells, animals, and humans.

Article Snippet: The following primary antibodies were used: Beclin1 (1∶1,000, Cell Signaling, #3495), pBeclin1 (S234 and S295, both 1∶1,000, Phosphosolutions, #p117-234 and #p117-295), Atg12 (1∶1,000, Cell Signaling, #2010), LC3B-II/I (1∶1,000, Cell Signaling, #2775), FLAG (1∶7,000, Rockland, 600-401-383), PI3K Class III (Vps34, 1∶1,000, Cell Signaling, #4263), FKBP51 (1∶1,000, Bethyl, A301-430A), Akt (1∶1,000, Cell Signaling, #4691), pAkt (Ser473 and T308, both 1∶1,000, Cell Signaling, #4058 and #9275), Actin (1∶5,000, Santa Cruz Biotechnology, sc-1616).

Techniques: Phospho-proteomics

Journal: Journal of Stroke

Article Title: Neuroprotective Effects of GV1001 in Animal Stroke Model and Neural Cells Subject to Oxygen-Glucose Deprivation/Reperfusion Injury

doi: 10.5853/jos.2021.00626

Figure Lengend Snippet: The effect of GV1001 on intracellular signaling proteins and cells. (A) Decreased levels of phosphorylated Akt (pAkt; Ser473), phospho-glycogen synthase kinase (pGSK-3β; Ser9), phosphorylated-extracellular signal-regulated kinase (pERK), B-cell lymphoma 2 (Bcl-2), and increased levels of Bcl-2 associated X (Bax) were noted in the peri-infarct regions of GV1001-treated rats compared to those of control rats. Administration of GV1001 (30 and 60 μM/kg) alleviated the changes. (B) Immunohistochemistry (IHC) staining showing increased pAkt (Ser473), pGSK-3β (Ser9), pERK, Bcl-2 levels and decreased Bax levels along with increased numbers of neuronal nuclei (NeuN)- or SRY-box transcription factor 2 (SOX2)-positive cells in GV1001-treated groups, scale bar: 50 μm. (C) IHC also revealed increased levels of nestin (a neuroectodermal stem cell marker), NeuN (a neuronal nuclear antigen), doublecortin (DCX; a neuronal differentiation marker), and SOX2 (a multipotent neural stem cell marker) in the peri-infarct region of GV1001-treated rats. Glial fibrillary acidic protein (GFAP; an astrocyte marker) expression was decreased in GV1001-treated rats, scale bar: 50 μm. (D) Levels of neurotoxic phenotype reactive astrocytes (uridine diphosphate glucuronosyltransferase 1A1 [UGT1A1] and FK506 binding protein 5 [FKBP5]) were increased and those of neuroprotective phenotype reactive astrocytes (sphingosine kinase type 1 [SPHK1]) were decreased in the peri-infarct area, but GV1001 significantly restored the expression of these markers. pIRS-1, phospho-insulin receptor substrate-1; DAPI, 4',6-diamidino-2-phenylindole. * P <0.05 (vs. sham group); † P <0.01 (vs. sham group); ‡ P <0.05 (vs. saline group); § P <0.01 (vs. saline group).

Article Snippet: The peri-infarct region in the control (sham and saline) group and the corresponding area in the GV1001-treated group, which were confirmed based on MRI, were used in Western blot analysis for phospho-insulin receptor substrate-1 (pIRS-1) (Ser636/639; 1:1,000, 2388, Cell Signaling Technology), phospho-phosphoinositide 3-kinase (pPI3K) p85 (Tyr458)/p55(Tyr199) (p85α PI3K; 1:1,000, 4228, Cell Signaling Technology), pAkt (Ser473), Akt (1:2,000, 9272, Cell Signaling Technology), pGSK-3β (1:1,000, 9336, Cell Signaling Technology), GSK-3β (1:2,000, sc-9166, Santa Cruz Biotechnology), pERK1/2 (Thr202/Tyr204), Bcl-2, Bax (1:1,000, 2772, Cell Signaling Technology), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1, 1:500, ab237810, Abcam), FK506 binding protein 5 (FKBP5, 0.4 μg/mL, NBP1-84676, Novus Biologicals, Centennial, CO, USA), sphingosine kinase type 1 (SPHK1, 1:1,000, ab71700, Abcam), and beta-tubulin (β-tubulin, 1:2,000, 2146, Cell Signaling Technology).

Techniques: Control, Immunohistochemistry, Marker, Expressing, Binding Assay, Saline

Journal: PLoS ONE

Article Title: Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding

doi: 10.1371/journal.pone.0137855

Figure Lengend Snippet: 40 most highly differentially regulated genes in HESCs treated by E 2 +MPA vs . E 2 alone according to whole genome microarray analysis using Illumina HumanHT–12 v4 expression BeadChip kit. All gene symbols were abbreviated according to GENEBANK standard nomenclature.

Article Snippet: The FKBP51 goat polyclonal antibody (Cat# AF4094) was obtained from R&D Systems, Minneapolis, MN.

Techniques: Microarray, Expressing, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding

doi: 10.1371/journal.pone.0137855

Figure Lengend Snippet: (A) q-PCR and (B) immunoblot analysis of FKBP51 levels in HESCs treated with estradiol (E 2 , 10 −8 M) ± progesterone (P 4 , 10 −7 M) or etonogestrel (ETO, 10 −7 M) or medroxyprogesterone acetate (MPA, 10 −7 M) for 6h or 24 h, respectively. Bars represent mean ± SEM (n = 3). * p<0 . 001 in E 2 +ETO vs . E 2 ; # p<0 . 001 in E 2 +MPA vs . E 2 .

Article Snippet: The FKBP51 goat polyclonal antibody (Cat# AF4094) was obtained from R&D Systems, Minneapolis, MN.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding

doi: 10.1371/journal.pone.0137855

Figure Lengend Snippet: (A_C) FKBP51 expression in paired endometrium of women pre- and post- DMPA administration. Immunoreactive FKBP51 in endometrial stromal (arrowheads) and glandular (arrow) cells pre- (A) and post- (B) DMPA use. HSCORE analysis of FKBP51 expression (C) in stromal and glandular cells. Bars represent mean ± SEM (n = 6). * p< 0 . 01 and # p<0 . 01 . Pre-DMPA: before DMPA use; Post-DMPA: 3 months DMPA use. (D-H) FKBP51 expression in endometria of OVX-GPs treated by MPA. Immunoreactivity for FKBP51 (brown) in stromal (arrowhead) and epithelial (arrows) cells of endometria of OVX-GPs treated with vehicle (D) or E 2 (E) or MPA (F) or E 2 +MPA (G) for 21 days. HSCORE analysis of FKBP51 immunoreactivity (H) in stromal and glandular cells. Bars represent mean ± SEM (n = 3 per treatment group). † p<0 . 001 in E 2 +MPA vs . E 2 or placebo (Pla); ѱ p<0 . 001 in E 2 +MPA vs . MPA or E 2 or Pla; ¥ in p<0 . 05 in MPA vs . E 2 or Pla; ‡ p<0 . 05 in MPA vs . E 2 and Pla. Original Magnification: A-G x40.

Article Snippet: The FKBP51 goat polyclonal antibody (Cat# AF4094) was obtained from R&D Systems, Minneapolis, MN.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding

doi: 10.1371/journal.pone.0137855

Figure Lengend Snippet: (A) Basal IL-1β mRNA levels in 10 −8 M estradiol (E) vs . E+ 10 −7 M MPA (EM) treated HESCs at 44 hrs post-transfection with either a control (Cont-V) or FKBP51 vector (FKBP51-V). Bars represent mean ± SEM (n = 4 with 3 replicates per treatment group). (B) In parallel incubations, thrombin- (Th-) induced IL-1β mRNA levels in E 2 +MPA treated HESCs at 44 hrs post-transfection with control or FKBP51 vector. Bars represent mean ± SEM (n = 2 and 3 replicates per treatment group). E: E 2 and EM: E 2 +MPA, * p<0 . 05 vs . EM treated in Cont-V transfected HESCs; ¥ p<0 . 05 vs . EM treatment in Cont-V transfected HESCs; † p<0 . 05 vs . EM; ‡ p<0 . 05 vs . EM+Th.

Article Snippet: The FKBP51 goat polyclonal antibody (Cat# AF4094) was obtained from R&D Systems, Minneapolis, MN.

Techniques: Transfection, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding

doi: 10.1371/journal.pone.0137855

Figure Lengend Snippet: LAPCs reduce endometrial blood flow causing hypoxia (HX) and generating reactive oxygen species (ROS), to directly damage blood vessels and enhance HEEC expression of angiopoietin (Ang)-2 and HESC expression of vascular endothelial growth factor (VEGF), interleukin (IL)-8 and matrix metalloproteinases (MMPs) while inhibiting HESC Ang–1 expression. The resultant inflammation and aberrant angiogenic stimulus leads to vessel damage causing local bleeding. The later delivers circulating factor VII to HESC membrane bound tissue factor (TF) which activates factor Xa to ultimately generate thrombin. The later exacerbates HX/ROS effects on endometrial angiogenesis and inflammation. In turn, HX and LAPCs trigger HEEC apoptosis by a paracrine manner. In addition, LAPCs also enhance FKBP51 expression in HESCs to inhibit progesterone receptor (PR; NR3C3) and glucocorticoid receptor (GR; NR3C1)-mediated transcription. In turn, the resultant PR and/or GR-mediated functional withdrawal reduces progestin and glucocorticoid mediated inhibition of endometrial inflammation, aberrant angiogenesis, thus contributing to AUB.

Article Snippet: The FKBP51 goat polyclonal antibody (Cat# AF4094) was obtained from R&D Systems, Minneapolis, MN.

Techniques: Expressing, Membrane, Functional Assay, Inhibition

Journal: PLoS ONE

Article Title: Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding

doi: 10.1371/journal.pone.0137855

Figure Lengend Snippet: 40 most highly differentially regulated genes in HESCs treated by E 2 +ETO vs . E 2 alone according to whole genome microarray analysis using Illumina HumanHT–12 v4 expression BeadChip kit. All gene symbols were abbreviated according to GENEBANK standard nomenclature.

Article Snippet: The FKBP51 goat polyclonal antibody (Cat# AF4094) was obtained from R&D Systems, Minneapolis, MN.

Techniques: Microarray, Expressing, Clinical Proteomics, Membrane

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

doi: 10.33594/000000230

Figure Lengend Snippet: Fig. 7. FKBP12 and FKBP51 protein expressions in Tacrolimus-, Sirolimus- and Everolimus-treated HepG2 (A and B, respectively) and Huh7 (C and D, respectively) cells. Treatments were administered at different concentrations (0, 10 nM, 10 µM, and 100 µM). The protein expression of FKBP12 and FKBP51 was evaluated by Western‐blot analysis as described in Material and Methods. Results are expressed as mean ± SEM, and blots are representative of four to six independent experiments. *p ≤ 0.05 between control and immunosuppressant‐treated cells. The groups with different letters (a, b, c or d) were significantly different (p ≤ 0.05).

Article Snippet: KG Navarro-Villarán et al.: Role of Immunosuppressant and FK506-Binding Protein Complex in Liver Cancer and Ser15P-p53 (#9284) obtained from Cell Signaling Technology (Danvers, Massachusetts, USA); LC3 (PM036) purchased from MBL International (Woburn, Massachusetts, USA); GADD153 (C/EBP homologous protein or CHOP) (sc-575), Beclin (sc-48341), p21 (sc-397) and p53 (sc-6243) obtained from Santa Cruz Biotechnology (Dallas, Texas, USA); Thr172P-Cdk4 (PA5-64482) obtained from ThermoFisher (Waltham, Massachusetts, USA); FKBP12 (Ref NB300-508) and FKBP38 (Ref NBP1-77909) obtained from Novus Biologicals (Centennial, Colorado, USA); and FKBP51 (Ref MAB4094) and FKBP52 (Ref MAB4095) obtained from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Expressing, Western Blot, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

doi: 10.33594/000000230

Figure Lengend Snippet: Fig. 9. Impact of FKBP51 downregulation on BrdU incorporation (A) and caspase-3 activity (B) in Tacrolimus-, Sirolimus- and Everolimus-treated HepG2 cells. The downregulation of FKBP51 was carried using siRNA technologies. Cell proliferation and apoptosis were determined using commercial BrdU incorporation and caspase‐3 activity assays respectively as described in Material and Methods. Results are expressed as mean ± SEM of six independent experiments. *p ≤ 0.05 and **p ≤ 0.01 between control and immunosuppressant‐ treated cells. The groups with different letters (a, b, c, d, e or f) were significantly different (p ≤ 0.05).

Article Snippet: KG Navarro-Villarán et al.: Role of Immunosuppressant and FK506-Binding Protein Complex in Liver Cancer and Ser15P-p53 (#9284) obtained from Cell Signaling Technology (Danvers, Massachusetts, USA); LC3 (PM036) purchased from MBL International (Woburn, Massachusetts, USA); GADD153 (C/EBP homologous protein or CHOP) (sc-575), Beclin (sc-48341), p21 (sc-397) and p53 (sc-6243) obtained from Santa Cruz Biotechnology (Dallas, Texas, USA); Thr172P-Cdk4 (PA5-64482) obtained from ThermoFisher (Waltham, Massachusetts, USA); FKBP12 (Ref NB300-508) and FKBP38 (Ref NBP1-77909) obtained from Novus Biologicals (Centennial, Colorado, USA); and FKBP51 (Ref MAB4094) and FKBP52 (Ref MAB4095) obtained from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: BrdU Incorporation Assay, Activity Assay, Control