Journal: The Journal of Biological Chemistry

Article Title: FK506-binding protein-like and FK506-binding protein 8 regulate dual leucine zipper kinase degradation and neuronal responses to axon injury

doi: 10.1016/j.jbc.2022.101647

Figure Lengend Snippet: FKBPL and FKBP8 induced lysosome-dependent DLK degradation. A , comparative analysis of relative expression levels in mouse L4,5 dorsal root ganglion (DRG) tissues, sciatic nerve tissues, and cultured embryonic DRG neurons ( , , , ). Red and blue circles indicate Illumina short-read sequencing and Oxford Nanopore direct RNA long-read sequencing, respectively. Circle sizes indicate relative levels of microarray data from cultured embryonic DRG neurons. B , Western blot analysis for the expression of DLK with FKBPs (N; null vector, L; FKBPL, 3; FKBP3, 4; FKBP4, 8; FKBP8, 12; FKBP12, 14; FKBP14, 15; FKBP15). The number indicates normalized relative intensity. Dual leucine zipper kinase and FLAG-epitope-tagged FKBP protein family members were expressed in HEK293T cells and subjected to SDS-PAGE. C , Western blot analysis for the immunoprecipitation of DLK with mouse (m) and human (h) FKBP4/8 that was overexpressed in HEK293T cells. Empty arrowhead , non-specific band; blue arrowhead , FKBP4; red arrowhead , FKBP8. D , Western blot analysis for the expression of DLK and FKBPL/4/8 expressed in HEK293T cells with or without bafilomycin A1 treatment. The numbers indicate the normalized relative intensity. E , Western blot analysis of DLK protein levels under Fkbp8 knockdown (sh Fkbp8 ) by lentiviral delivery in primary cultured embryonic DRG neurons. The numbers indicate the normalized relative intensity. F , statistical analysis of ( E ) (FC, fold change; n = 3 for each condition; ∗ p < 0.05 by t test; mean ± S.E.M.). DLK, dual leucine zipper kinase.

Article Snippet: An expression plasmid for full-length Myc-DDK-tagged Mouse Fkbp3 (MR202616), Fkbp4 (MR227193), Fkbp8 (MR220865), Fkbp12 (MR200405), Fkbp14 (MR202290), and Fkbp15 (MR220579) were purchased from Origene.

Techniques: Expressing, Cell Culture, Sequencing, Microarray, Western Blot, Plasmid Preparation, FLAG-tag, SDS Page, Immunoprecipitation, Knockdown

Journal: Cell death & disease

Article Title: Melatonin-mediated FKBP4 downregulation protects against stress-induced neuronal mitochondria dysfunctions by blocking nuclear translocation of GR.

doi: 10.1038/s41419-023-05676-5

Figure Lengend Snippet: Fig. 3 Melatonin downregulates FKBP4, but is not related to phosphorylation of GR. A–C, F SH-SY5Y cells were treated with melatonin (1 μM) for 30 min and then with cortisol (1 μM) for 24 h. A The expression of p-GR and GR were detected by western blot. Loading control is β-actin. n = 5. B The expression of GR protein in subcellular fraction samples was detected by western blotting. Lamin A/C and α-tubulin were used as a nuclear and cytosolic loading control, respectively. n = 5. C The cells were immunostained with GR (green) and DAPI (blue). Scale bars, 10 μm (magnification, ×1000). n = 5. D, G Mice were injected with melatonin (10 mg/kg) and then with corticosterone (10 mg/kg) for 7 days. D Slide samples for immunohistochemistry were immunostained with GR (green) and DAPI (blue). Scale bars, 140 μm (magnification, ×100). n = 5. E SH-SY5Y cells were treated with melatonin for 30 min and then with cortisol for 12 h. The mRNA expression of regulatory proteins related to cytosolic GR complex, dynein complex, and NPC were analyzed by real time PCR. n = 5. F, G FKBP4 was detected by western blot. Loading control is β-actin. n = 5. All blots and immunofluorescence images are representative. The representative images were acquired by SRRF imaging system. All data are presented as a mean ± S.E.M. *p < 0.05 versus control, #p < 0.05 versus cortisol or corticosterone.

Article Snippet: MT1 (NBP1-71113), FKBP4 (NB110-96874), FKBP5 (NBP1-84676), LC3 (NB100-2220) and NIX (NBP1-88558) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Control, Injection, Immunohistochemistry, Real-time Polymerase Chain Reaction, Imaging

Journal: Cell death & disease

Article Title: Melatonin-mediated FKBP4 downregulation protects against stress-induced neuronal mitochondria dysfunctions by blocking nuclear translocation of GR.

doi: 10.1038/s41419-023-05676-5

Figure Lengend Snippet: Fig. 4 Melatonin-mediated FKBP4 downregulation blocks dynein-dependent GR nuclear translocation. A, B, D, F, G SH-SY5Y cells were treated with melatonin (1 μM) for 30 min and then with cortisol (1 μM) for 24 h. A GR was co-immunoprecipitated with FKBP4 and FKBP5. The level of FKBP4 protein in immunoprecipitated samples was quantified. n = 5. B The interaction between GR and FKBP4 was investigated via proximity ligation assay. Scale bars, 10 μm (magnification, ×1000). n = 5. C, E Mice were injected with melatonin (10 mg/kg) and then with corticosterone (10 mg/kg) for 7 days. C Slide samples for immunohistochemistry were immunostained with GR (green), FKBP4 (red) and DAPI (blue). Scale bars, 140 μm (magnification, ×100). n = 5. D The cells were immunostained with GR (red), dynein (green) and DAPI (blue). Scale bars, 10 μm (magnification, ×1000). n = 5. E Slide samples for immunohistochemistry were immunostained with dynein (red), GR (green), and DAPI (blue). Scale bars, 140 μm (magnification, ×100). n = 5. F The cells were immunostained with GR (green), NUP62 (red), and DAPI (blue). Scale bars, 10 μm (magnification, ×1000). n = 5. G The cells were immunostained with GR (green), importin β (red), and DAPI (blue). Scale bars, 10 μm (magnification, ×1000). n = 5. All blots and immunofluorescence images are representative. The representative images were acquired by SRRF imaging system. All data are presented as a mean ± S.E.M. *p < 0.05 versus control, #p < 0.05 versus cortisol or corticosterone.

Article Snippet: MT1 (NBP1-71113), FKBP4 (NB110-96874), FKBP5 (NBP1-84676), LC3 (NB100-2220) and NIX (NBP1-88558) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Translocation Assay, Immunoprecipitation, Proximity Ligation Assay, Injection, Immunohistochemistry, Imaging, Control

Journal: Cell death & disease

Article Title: Melatonin-mediated FKBP4 downregulation protects against stress-induced neuronal mitochondria dysfunctions by blocking nuclear translocation of GR.

doi: 10.1038/s41419-023-05676-5

Figure Lengend Snippet: Fig. 5 Melatonin upregulates DNMT1 via MT1/ERK/c-MYC axis. A The cells were treated with actinomycin D (500 ng/ml) 30 min before treating melatonin for 12 h. The FKBP52 mRNA expression was analyzed by real-time PCR. n = 5. B, K SH-SY5Y cells were treated with melatonin (1 μM) for 30 min and then with cortisol (1 μM) for 12 h. B The mRNA expression of epigenetic-regulated genes was analyzed by real time PCR. n = 5. C SH-SY5Y cells were treated with melatonin for 30 min and then with cortisol for 24 h. D Mice were injected with melatonin (10 mg/kg) and then with corticosterone (10 mg/kg) for 7 days. C, D DNMT1 expression was detected by western blot. Loading control is β-actin. n = 5. E, F, H SH-SY5Y cells were treated with melatonin for 12 h. E The MT1 and MT2 mRNA expression were analyzed by real time PCR. n = 5. F MT1 and MT2 levels were detected by western blot. Loading control is β-actin. n = 5. G The cells were transfected with NT or MT1 siRNA 24 h before melatonin treatment for 24 h. FKBP4 levels were detected by western blot. Loading control is β-actin. n = 5. H The cells were immunostained with MT1 (green), Gαq (red) and DAPI (blue). Scale bars, 10 μm (magnification, ×1000). n = 5. I The cells were transfected with NT or MT1 siRNA 24 h before melatonin treatment for 12 h. The p-ERK and ERK levels were investigated by western blot. Loading control is β-actin. n = 5. J PD98059 (50 μM) was treated 30 min before melatonin treatment for 12 h. The p-c-MYC and c-MYC levels were detected by western blot. Loading control is β-actin. n = 5. K The cells were immunostained with c-MYC (green) and DAPI (blue). Scale bars, 10 μm (magnification, ×1000). n = 5. All blots and immunofluorescence images are representative. The representative images were acquired by SRRF imaging system. All data are presented as a mean ± S.E.M. *p < 0.05 versus control or control + NT siRNA, #p < 0.05 versus melatonin or melatonin + NT siRNA.

Article Snippet: MT1 (NBP1-71113), FKBP4 (NB110-96874), FKBP5 (NBP1-84676), LC3 (NB100-2220) and NIX (NBP1-88558) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Injection, Western Blot, Control, Transfection, Imaging

Journal: Cell death & disease

Article Title: Melatonin-mediated FKBP4 downregulation protects against stress-induced neuronal mitochondria dysfunctions by blocking nuclear translocation of GR.

doi: 10.1038/s41419-023-05676-5

Figure Lengend Snippet: Fig. 7 The schematic image is presented. Glucocorticoid induces nuclear translocation of GR and then represses NIX transcription. Melatonin downregulates FKBP4 expression by increasing DNMT1 levels to suppress nuclear translocation of GR, which then restores NIX-mediated mitophagy and neuronal cell survival.

Article Snippet: MT1 (NBP1-71113), FKBP4 (NB110-96874), FKBP5 (NBP1-84676), LC3 (NB100-2220) and NIX (NBP1-88558) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Translocation Assay, Expressing

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Physiological role for the cochaperone FKBP52 in androgen receptor signaling.

doi: 10.1210/me.2005-0071

Figure Lengend Snippet: Fig. 1. Targeting Strategies and Verification of Gene Dele- tion A, Partial restriction map of the wild-type (wt) and mutant (mut) Fkbp4 alleles and schematic depicting the targeting strategy used to delete the entire Fkbp4 coding region. The 10 coding exons in the wt allele are depicted as solid boxes; the final exon contains both coding and noncoding (open box) sequences. The neomycin resistance cassette (neo) is indicated as are restriction enzyme sites for BamHI (B), EcoRI (E), HindIII (H), KpnI (K), PstI (P), and SacI (S). The bar near the 5 end of either allele indicates the probe used for Southern blots. Short, bold arrows on the 3 half indicate PCR primers used for genotyping. B, Southern blot analysis of mouse tail genomic DNA reveals disruption of Fkbp4. Digestion of genomic DNA with EcoRI generated a 17-kb wild-type (/) and a 9-kb targeted (/) fragment, as detected by hybrid- ization with the 5 probe. C, Western blot analysis of thymic cytosol confirmed the absence of FKBP52 protein in homozy- gous mutant tissue.

Article Snippet: No other uses without permission. . All rights reserved. available human multiple tissue blot (Human Normal Tissue Blot IV; ProSci, Poway, CA) was probed with antibodies recognizing FKBP52 or GAPDH.

Techniques: Mutagenesis, Southern Blot, Disruption, Generated, Western Blot

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Physiological role for the cochaperone FKBP52 in androgen receptor signaling.

doi: 10.1210/me.2005-0071

Figure Lengend Snippet: Fig. 3. Immunohistochemistry of Murine Testis and Anterior Prostate Testis (A–F) and anterior prostate (G–L) were collected from 8- to 10-wk adult wild-type (A–C; G–I) or 52KO (D–F; J–L) males. Although anterior prostate is often absent in 52KO males, tissue was collected from an animal that retained some anterior pros- tate development. Consecutive tissue sections were immuno- stained (red stain) with prebleed serum (A, D, G, and J), affinity- purified anti-FKBP52 (B, E, H, and K), or anti-AR PG-21 (C, F, I, and L), and all sections were counterstained with hematoxylin (blue stain). Specific FKBP52 staining is readily detected in the cytoplasm of most cell types in the wild-type testis (B) and in epithelial cells of the wild-type anterior prostate (H), but FKBP52 staining is absent, as expected, in corresponding tissues from 52KO mice (E and K). Specific AR staining in either wild-type (C) or 52KO testis (F), was detected in nuclei of Sertoli cells (Sc), Leydig cells (Lc), and peritubular myoid cells (pmc) of stage VIII seminiferous tubules. Different generations of spermatogenic cells, including mature spermatids (sp), can be seen in both wild-type and 52KO seminiferous tubules (A–F). Specific AR staining was also observed in the nuclei of ductal epithelial cells from the anterior prostate of either wild-type (I) or 52KO (L) mice.

Article Snippet: No other uses without permission. . All rights reserved. available human multiple tissue blot (Human Normal Tissue Blot IV; ProSci, Poway, CA) was probed with antibodies recognizing FKBP52 or GAPDH.

Techniques: Immunohistochemistry, Staining, Affinity Purification

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Physiological role for the cochaperone FKBP52 in androgen receptor signaling.

doi: 10.1210/me.2005-0071

Figure Lengend Snippet: Fig. 5. FKBP52 Is Expressed in Human Reproductive Organs A, Western blot analysis of extracts (15 g total protein/ lane) from human reproductive tissues established that FKBP52 is present in all extracts, but at a relatively low level in placenta. Samples were immunostained for GAPDH as an internal reference. B, Waste samples of prostate tissue were obtained from adult males surgically treated for benign pros- tatic hyperplasia. Tissues were fixed, embedded, and sec- tioned before staining protocols. A representative section was histologically stained with hematoxylin and eosin (upper left panel), and consecutive sections were immunostained with preimmune antiserum or antibody specific for AR or FKBP52, as indicated. The strongest staining for AR and FKBP52 is in ductal epithelial cells; AR staining is mostly localized to nuclei whereas FKBP52 staining is predominantly cytoplasmic. The example shown is typical of staining pat- terns for prostate specimens from three patients.

Article Snippet: No other uses without permission. . All rights reserved. available human multiple tissue blot (Human Normal Tissue Blot IV; ProSci, Poway, CA) was probed with antibodies recognizing FKBP52 or GAPDH.

Techniques: Western Blot, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Physiological role for the cochaperone FKBP52 in androgen receptor signaling.

doi: 10.1210/me.2005-0071

Figure Lengend Snippet: Fig. 6. In Vitro and Cellular Interactions of FKBP52 and AR A, Radiolabeled human AR was incubated in rabbit reticulocyte lysate under conditions that promote maximal assembly of receptor/chaperone complexes. The assembly mixtures contained no added compound or compounds known to influence receptor/chaperone complexes. DHT (100 nM) would be expected to promote dissociation of receptor/chaperone complexes. Geldanamycin (GA, 20 g/ml), an Hsp90 inhibitor, would be expected to arrest assembly of chaperone complexes before the mature stage at which FKBP cochaperones enter the complex. Finally, FK506 (10 M) is an FKBP-specific peptidylprolyl isomerase inhibitor. The AR assembly mixtures were coimmunoprecipitated using an immunoaffinity resin specific for FKBP52 or a negative control (Ctrl.) resin, as indicated above the gel lanes. Samples were separated by SDS-PAGE, Coomassie-stained to visualize total proteins (large upper panel), and autoradiographed to detect radiolabeled AR. Note that AR specifically copre- cipitates with FKBP52 but is largely displaced by each of the treatments tested. The final lane is an aliquot of the AR synthesis mixture that illustrates the input for each assembly sample. B, A yeast AR/lacZ reporter strain was transformed with an empty vector (open circle) or plasmids expressing human FKBP51 (closed circles) or FKBP52 (closed squares). DHT dose-response curves were generated for hormone-induced -galactosidase expression. C, The yeast AR/lacZ reporter strain was transformed with an empty plasmid (vector) or a plasmid expressing human FKBP52, FKBP51, PP5, an FKBP52 mutant that does not bind Hsp90 (52-K354A), or a mutant deficient in PPIase activity (52-FD67DV). Reporter activity was measured in yeast extracts (n 3) after induction with 10 nM DHT. For reference, the level of hormone-induced reporter expression observed in the background strain (vector) is 50-fold greater than reporter expression in the absence of hormone (not shown). The inset panel illustrates expression levels of AR, FKBP52 forms, FKBP51, PP5, and the endogenous ribosomal subunit L3 (left-hand labels) in experi- mental strains (labeled above the lanes) as determined by Western blots of yeast extracts (inset panel). D, The AR/lacZ reporter strain containing an empty vector or plasmid expressing FKBP52 was induced with 10 nM DHT after a pretreatment (solid bars) or not (open bars) with 10 M FK506.

Article Snippet: No other uses without permission. . All rights reserved. available human multiple tissue blot (Human Normal Tissue Blot IV; ProSci, Poway, CA) was probed with antibodies recognizing FKBP52 or GAPDH.

Techniques: In Vitro, Incubation, Negative Control, SDS Page, Staining, Transformation Assay, Plasmid Preparation, Expressing, Generated, Mutagenesis, Activity Assay, Labeling, Western Blot

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Physiological role for the cochaperone FKBP52 in androgen receptor signaling.

doi: 10.1210/me.2005-0071

Figure Lengend Snippet: Fig. 7. Effect of FKBP52 Knockdown on AR Transactivation in Human Cells HeLa-AR cells were stably infected with virus expressing a scramble shRNA (Control) or an shRNA targeting FKBP52 expression (52KD). Cell lines were cotransfected with a lucif- erase reporter plasmid plus an empty plasmid (vect) or plas- mid expressing an FKBP52 silent mutant that escapes knockdown (p52). A, Extracts from each of the experimental lines were Western blotted for AR, FKBP52, or GAPDH, the latter as an internal loading control. B, Replicate cell samples (n 3) were treated or not with DHT (10 nM) for 24 h and assayed for induced luciferase activity. Overexpression of exogenous FKBP52 in Control cells heightened reporter ac- tivity (Control/p52/) compared with cells having endoge- nous FKBP52 only (Control/vect/). Hormone-induced re- porter activity was lowered in cells with reduced FKBP52 expression (52KD/vect/) compared with Control cells, and reporter expression could be partially restored by expression of exogenous FKBP52 (52KD/p52/). C, Specific hormone- binding measurements (n 3 for each data point) were generated for HeLa-AR Control (solid circles) or 52KD (open circles). The two binding curves closely overlap, indicating no difference in AR hormone binding affinity. D, Hormone dose- response curves were generated for HeLa-AR Control (closed circles) or 52KD (open circles) cells transfected with a luciferase reporter plasmid. Knockdown of FKBP52 expres- sion decreased the maximal response to hormone.

Article Snippet: No other uses without permission. . All rights reserved. available human multiple tissue blot (Human Normal Tissue Blot IV; ProSci, Poway, CA) was probed with antibodies recognizing FKBP52 or GAPDH.

Techniques: Knockdown, Stable Transfection, Infection, Virus, Expressing, shRNA, Control, Plasmid Preparation, Mutagenesis, Western Blot, Luciferase, Activity Assay, Over Expression, Binding Assay, Generated, Transfection

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Physiological role for the cochaperone FKBP52 in androgen receptor signaling.

doi: 10.1210/me.2005-0071

Figure Lengend Snippet: Fig. 8. Response of Mutant AR to FKBP52 A, Yeast reporter strains expressed either wild-type AR (wtAR) or an AR point mutant (AR-P723S). Experimental strains coexpressed human FKBP51, FKBP52, mutant forms of FKBP52, Hip, or an empty vector, as indicated. Replicates (n 3) of each experimental strain were treated with 10 nM DHT and assayed for induced reporter activity. B, Extracts were prepared from experimental strains and assayed by Western blots for levels of mutant AR, FKBP, Hip, and endogenous L3. The FKBP blot was immunostained with a mixture of anti-FKBP51 and anti-FKBP52 antibodies to detect either protein. C, DHT dose-response curves were generated for AR-P723S reporter strains that coexpress either FKBP51 (open circles) or FKBP52 (closed circles). D, Cocrystal structure of AR ligand binding domain illustrating the positions of Pro723 (green), helices H3, H4, and H12 (red), hormonal ligand (orange), and peptide from the coactivator TIF2 (transcriptional intermediary factor 2) (blue). Adapted from Brookhaven databank file 1XQ2.

Article Snippet: No other uses without permission. . All rights reserved. available human multiple tissue blot (Human Normal Tissue Blot IV; ProSci, Poway, CA) was probed with antibodies recognizing FKBP52 or GAPDH.

Techniques: Mutagenesis, Plasmid Preparation, Activity Assay, Western Blot, Generated, Ligand Binding Assay

Journal: The American journal of pathology

Article Title: Enhanced Human Decidual Cell-Expressed FKBP51 May Promote Labor-Related Functional Progesterone Withdrawal.

doi: 10.1016/j.ajpath.2015.05.014

Figure Lengend Snippet: Figure 3 FKBP51 and FKBP52 mRNA expression in term decidual cell (DC) monolayers. Confluent leukocyte-free term DCs from decidua parietalis were primed for 7 days with 108 mol/L estradiol (E2), E2 þ 107 mol/L medroxyprogesterone acetate (MPA), E2 þ dexamethasone (Dex), or E2 þ Organon (Org) 2058, then incubated with corresponding steroids in DM Q27 for 6 hours. Quantitative real-time RT-PCR for FKBP51 (A) and FKBP52 (B) mRNA levels for term DCs measured and normalized to b-actin. Data represent means SEM (A and B). n Z 4 (A and B). ***P < 0.001 E2 þ MPA or E2 þ Dex versus E2 alone. DM, defined media.

Article Snippet: FKBP51 and FKBP52 Overexpression To overexpress FKBP51 and FKBP52 in term DCs, expression vector containing the full-length FKBP51 gene open reading frame (pCMV6-FKBP51 or pCMV6FKBP52) and empty vector (pCMV6) were purchased from Origene Inc. (Rockville, MD).

Techniques: Expressing, Incubation, Quantitative RT-PCR

Journal: The American journal of pathology

Article Title: Enhanced Human Decidual Cell-Expressed FKBP51 May Promote Labor-Related Functional Progesterone Withdrawal.

doi: 10.1016/j.ajpath.2015.05.014

Figure Lengend Snippet: Figure 2 Comparison of FKBP51 and FKBP52 mRNA levels in term decidual cells (DCs), cytotrophoblasts (CYTs), and syncytiotrophoblasts (SCTs) by quantitative real-time PCR. FKBP51 (A) and FKBP52 (B) mRNA levels were determined and normalized to b-actin in term DCs (TDCs), CYTs, and SCTs after 6 hours of incubation with control (Cont) or 107 mol/L Dex (þDex). Data represent means SEM from three replicates (A and B). n Z 3 (A and B). *P < 0.05 Cont CYT or SCT versus TDC, Dex-treated CYT or SCT versus Dex-treated TDC, Cont CYT versus SCT, Dex-treated CYT versus Dex- treated SCT; ***P < 0.001 Dex-treated TDC versus Cont TDC.

Article Snippet: FKBP51 and FKBP52 Overexpression To overexpress FKBP51 and FKBP52 in term DCs, expression vector containing the full-length FKBP51 gene open reading frame (pCMV6-FKBP51 or pCMV6FKBP52) and empty vector (pCMV6) were purchased from Origene Inc. (Rockville, MD).

Techniques: Comparison, Real-time Polymerase Chain Reaction, Incubation, Control

Journal: The American journal of pathology

Article Title: Enhanced Human Decidual Cell-Expressed FKBP51 May Promote Labor-Related Functional Progesterone Withdrawal.

doi: 10.1016/j.ajpath.2015.05.014

Figure Lengend Snippet: Figure 4 FKBP51, FKBP52, progesterone re- ceptor (PR), and glucocorticoid receptor (GR) protein expression in nuclear extracts of decidual cell (DC) monolayers. Confluent leukocyte-free DCs were primed for 7 days with vehicle [control (C)], 108 mol/L estradiol (E2), 107 mol/L medrox- yprogesterone acetate (MPA), 107 mol/L dexa- methasone (Dex), 107 mol/L Organon (Org) 2058, E2 þ MPA, E2 þ Dex, or E2 þ Org2058, then incubated for 24 hours with corresponding steroids in Q28 DM. Immunoblot readings were normalized to TATA-binding protein (TBP) as loading control. A: Representative immunoblotting displaying bands for PR-A and PR-B isoforms, GR, FKBP51, FKBP52, and TBP. Graphs display immunoblot densitometry readings obtained from experimental incubations of term DC cultures for FKBP51/TBP (B), PR/TBP (C), and GR/TBP (D). Data represent means SEM (C and D). n Z 3 (C and D). *P < 0.05 for E2 þ MPA or E2 þ Org versus E2 alone or E2 þ MPA versus E2 þ Org; **P < 0.01 for MPA or Dex versus C or E2 þ MPA or E2 þ Dex versus E2 alone; ***P < 0.001 for E2 versus C; yP < 0.05 for Dex versus C, or E2 þ Dex versus E2 alone. DM, defined media. Q29

Article Snippet: FKBP51 and FKBP52 Overexpression To overexpress FKBP51 and FKBP52 in term DCs, expression vector containing the full-length FKBP51 gene open reading frame (pCMV6-FKBP51 or pCMV6FKBP52) and empty vector (pCMV6) were purchased from Origene Inc. (Rockville, MD).

Techniques: Expressing, Control, Incubation, Western Blot, Binding Assay

Journal: The American journal of pathology

Article Title: Enhanced Human Decidual Cell-Expressed FKBP51 May Promote Labor-Related Functional Progesterone Withdrawal.

doi: 10.1016/j.ajpath.2015.05.014

Figure Lengend Snippet: Figure 5 FKBP51, FKBP52, progesterone re- ceptor (PR), and glucocorticoid receptor (GR) protein expression in cytosolic extracts of decidual cell (DC) monolayers. Cytosolic extracts from the experiments described in Figure 4 were analyzed by immunoblotting, and results were normalized to b-actin as loading control. A: Representative immunoblotting displaying bands for PR-A and PR-B isoforms, GR, FKBP51, FKBP52, and b-actin. Graphs display densitometric analysis of immuno- blot readings obtained from experimental in- cubations of term DC cultures isolated from women for FKBP51/b-actin (B), PR/b-actin (C), and GR/b-actin (D). Data represent means SEM (BeD). n Z 3 (BeD). *P < 0.05 for medroxy- progesterone acetate (MPA) or dexamethasone (Dex) versus control (C) and estradiol (E2) þ MPA or E2 þ Dex versus E2 alone; ***P < 0.001 for Dex versus C; yP < 0.05 for MPA or Organon (Org) versus C, or E2 þ MPA, E2 þ Dex, or E2 þ Org versus E2 alone; zP < 0.05 E2 þ Dex versus E2 alone. Q30

Article Snippet: FKBP51 and FKBP52 Overexpression To overexpress FKBP51 and FKBP52 in term DCs, expression vector containing the full-length FKBP51 gene open reading frame (pCMV6-FKBP51 or pCMV6FKBP52) and empty vector (pCMV6) were purchased from Origene Inc. (Rockville, MD).

Techniques: Expressing, Western Blot, Control, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Early Life Stress Alters Expression of Glucocorticoid Stress Response Genes and Trophic Factor Transcripts in the Rodent Basal Ganglia

doi: 10.3390/ijms23105333

Figure Lengend Snippet: Simplified diagram of the glucocorticoid receptor (GR) stress signalling pathway. GR (unfolded) is unable to bind cortisol when bound to Bag1, and release of Bag1 allows GR to bind cortisol with low affinity, whereas Fkbp51 (encoded by Fkbp5 ) and p23 (encoded by Ptges3 ) are involved in increasing GR affinity to cortisol by stabilizing the GR heterocomplex into a high affinity state. Furthermore, Fkbp52 (encoded by Fkbp4 ) dislocates Fkbp51 and facilitates nuclear translocation of the cortisol-bound GR heterocomplex into the nucleus to activate or repress target genes. Thus, higher Fkbp51 would promote GR retention in the cytoplasm, rendering target genes less responsive to stress [ , , , ]. GR = glucocorticoid receptor; Hsp = heat shock protein.

Article Snippet: The probesets used were: Nr3c1 (GR)-Rn00561369_m1; Fkbp5 -Rn01768371_m1; Fkbp4- Rn01459299_m1; Ptges3 -Rn01529546_m1; Bag1-Rn01439834_m1; Bdnf I- Rn01484924_m1; Bdnf IIA -Rn00560868_m1; Bdnf IIC -Rn01484925_m1; Bdnf III -Rn04230563_m1; Bdnf IV -Rn01484927_m1; Bdnf VI -Rn01484928_m1.

Techniques: Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: Early Life Stress Alters Expression of Glucocorticoid Stress Response Genes and Trophic Factor Transcripts in the Rodent Basal Ganglia

doi: 10.3390/ijms23105333

Figure Lengend Snippet: Two-way ANOVA showing main effects of early life stress and sex, as well as interactions between early life stress and sex, on stress signalling-related transcripts in the substantia nigra, VTA, dorsal striatum and ventral striatum of 22 control and 24 maternally separated rodents. Asterisks (*) indicate genes whose log-transformed data were used for statistical analysis. VTA = ventral tegmental area; GR = glucocorticoid receptor. Bold type indicates statistically significant effects.

Article Snippet: The probesets used were: Nr3c1 (GR)-Rn00561369_m1; Fkbp5 -Rn01768371_m1; Fkbp4- Rn01459299_m1; Ptges3 -Rn01529546_m1; Bag1-Rn01439834_m1; Bdnf I- Rn01484924_m1; Bdnf IIA -Rn00560868_m1; Bdnf IIC -Rn01484925_m1; Bdnf III -Rn04230563_m1; Bdnf IV -Rn01484927_m1; Bdnf VI -Rn01484928_m1.

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Early Life Stress Alters Expression of Glucocorticoid Stress Response Genes and Trophic Factor Transcripts in the Rodent Basal Ganglia

doi: 10.3390/ijms23105333

Figure Lengend Snippet: Differences between females and males in stress-related transcript levels and effects of early life stress. ( A ) Females had significantly higher Fkbp4 mRNA levels in the substantia nigra and dorsal striatum than males; ( B ) female control rats had higher Fkbp5 mRNA levels in the substantia nigra than male control rats. They also had higher Fkbp5 mRNA levels than maternally separated female rats; ( C ) maternally separated male rats had lower Nr3c1 (GR) mRNA levels in the VTA than male control rats; ( D ) maternally separated male rats had lower Bag1 mRNA levels in the VTA than male control rats. Each data point represents a single animal. Filled squares and hollow diamonds represent female and male animals, respectively. Horizontal lines depict group means. * p < 0.05, ** p < 0.005, *** p < 0.0005.

Article Snippet: The probesets used were: Nr3c1 (GR)-Rn00561369_m1; Fkbp5 -Rn01768371_m1; Fkbp4- Rn01459299_m1; Ptges3 -Rn01529546_m1; Bag1-Rn01439834_m1; Bdnf I- Rn01484924_m1; Bdnf IIA -Rn00560868_m1; Bdnf IIC -Rn01484925_m1; Bdnf III -Rn04230563_m1; Bdnf IV -Rn01484927_m1; Bdnf VI -Rn01484928_m1.

Techniques: Control