PA1-511A

antibody from Invitrogen Antibodies

Targeting: NR3C1 GR, GRL

Provider product page for PA1-511A

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [111]
Comments [0]
Validations
Immunocytochemistry [3]
Immunohistochemistry [2]
Other assay [18]

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Product number: PA1-511A - Provider product page
Provider: Invitrogen Antibodies
Product name: Glucocorticoid Receptor Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-511A detects glucocorticoid receptor (GR) from human, mouse and rat tissues. For reconstitution, centrifuge the vial briefly, then add 50 µL PBS to achieve a final antibody concentration of 1 mg/mL. PA1-511A has been successfully used in Western blot, immunocytochemistry, immunofluorescence, and immunohistochemistry procedures. By Western blot, this antibody detects an ~97 kDa protein representing GR from rat liver extract. Immunocytochemical staining of GR in HeLa cells using PA1-511A results in cytoplasmic staining in the absence of ligand, and nuclear staining after hormone administration. The PA1-511A immunogen is a synthetic peptide corresponding to residues D(346) Q K P I F N V I P P I P V G S E N W N R C(367) from human GR. The PA1-511A immunizing peptide (Cat. # PEP-001) is available for use in neutralization and control experiments.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 50 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

NR3C1 protein structure - PA1-511A shown in red.

Supportive validation

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Experimental details: Immunofluorescent analysis of Glucocorticoid Receptor using Glucocorticoid Receptor Polyclonal Antibody (Product # PA1-511A) shows staining in Hela Cells. Glucocorticoid Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Glucocorticoid Receptor (Product # PA1-511A) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of Glucocorticoid Receptor using Glucocorticoid Receptor Polyclonal Antibody (Product # PA1-511A) shows staining in NIH-3T3 Cells. Glucocorticoid Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Glucocorticoid Receptor (Product # PA1-511A) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of Glucocorticoid Receptor using Glucocorticoid Receptor Polyclonal Antibody (Product # PA1-511A) shows staining in U251 Cells. Glucocorticoid Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Glucocorticoid Receptor (Product # PA1-511A) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunohistochemistry was performed on normal deparaffinized human Tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a rabbit polyclonal antibody recognizing Glucocorticoid Receptor (Product # PA1-511A) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized human Cervical carcinoma tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a rabbit polyclonal antibody recognizing Glucocorticoid Receptor (Product # PA1-511A) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

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Supportive validation

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Experimental details: Figure 7 Association of CREB binding sites with additional transcription factor occupancy at fasting-responsive genes. CREB sites were separated into 3 groups: 285 sites associated with genes up-regulated in fasted state (red), 321 sites associated with genes down-regulated in fasted state (green), 6,018 sites associated with genes that did not change (gray). A-F) Average ChIP-seq profiles around each group of CREB binding sites are shown for CREB ( A ), CEBPB ( B ), NR3C1/GR ( C ), FOXA2 ( D ), PPARA ( E ), and CTCF ( F ). Corresponding distributions of peak heights are shown as box plots in Additional file 1 : Figure S7 and tabulated in Additional file 3 . G ) Percentage of sites in each group containing the indicated motifs within +/- 250 bp of peak center. *p-value < 0.001 by Fisher's exact test compared to gray bar.

Supportive validation

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Experimental details: Figure 11 Kidney biopsies from normal kidney (control) and ccRCC patients were stained with anti-NNMT and anti-NR3C1 antibodies In the patients with confirmed ccRCC, serial sections showed stronger NNMT and NR3C1 immunoreactivity as compared to the controls.

Supportive validation

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Experimental details: Figure 5 RU486 directly regulate PTL transcription through nuclear receptor GR (A) Expression of Progesterone Receptor (PR) and glucocorticoid receptor (GR) in pancreas was examined by immuno-staining in frozen samples after cryosection. Differential Interference Contrast (DIC) microscope image from same field was shown on right. Arrows points to the positive staining of GR in the nucleus (in red). Scale bars for A and (B) 5um. (B) Acute DXMS administration (i.p.injected at 100mg/kg, 8 h) in mice promotes transcription of PTL and amylase in pancreas. n = 4 to 5 for each condition. (C) Schematics, top: two conserved GRE (GR Response Element) motif in promoter region of PTL (both in mice and rat) was shown. Bottom: Strategy of luciferase reporter assay to evaluate the GRE motif in PTL promoter region. (D) qPCR experiments shown that PTL promoter region are highly enriched in anti-GR ChIP samples from mice pancreas. (E) Incubation of DXMS (100nM, 24 h) promotes PTL and amylase transcription in AR42J cell lines, and this effect can be suppressed by coincubation of RU486. (F) The role of GRE motif in PTL promoter in response to DXMS was monitored in AR42J cell lines transfected with indicated luciferase reporters. These reporters contain luciferase fused with PTL promoter bearing wild type GRE(GRE WT luc) or deleted GRE (GRE Del luc).

Supportive validation

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Experimental details: Fig. 4 HNF4A, GR, STAT3, and STAT5 binding at enhancer elements. a Genome-wide coverage plots at all, SHARED, IRI-decreased and IRI-increased enhancers. The coverage for HNF4A and GR decreased in SHARED and IRI-decreased enhancer group after IRI. In contrast, STAT3 shows increased coverage at IRI-increased enhancers. STAT5 peak height at enhancer elements is unchanged after kidney injury. b Representative examples of transcription factor binding at enhancer sites in kidney epithelia cells. The Slc34a1 and Junb genomic locus are shown for HNF4A, GR, STAT3, and STAT5 binding together with H3K4me3 and H3K27ac in the SHAM (left) and IRI (right) condition. Enhancer elements are indicated by gray bars.

Supportive validation

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Experimental details: Figure 4 The effects of a fructose-rich diet on the level of GR, lipin-1 PEPCK, and G6Pase in the liver. Groups: control (C), fructose-fed (F). ( a ) GR protein level in hepatic cytosols and nucleosols, and ( b ) lipin-1 protein level in hepatic whole cell extracts, were measured by Western blot. Relative integrated optical density of the immunoreactive bands was assessed by ImageQuant software, normalized to equal load controls and expressed as fold of the control. The levels of PEPCK ( c ) and G6Pase ( d ) mRNA were determined by qPCR and expressed as fold of the control. The values represent the means +- SEM ( n = 9). Statistical significance of differences between experimental groups: * p < 0.05.

Supportive validation

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Experimental details: Fig. 1 Chromatin structures and activity of the Csn1s2a/b locus. a mRNA levels were measured by RNA-seq at day 18 of pregnancy (p18), day one of lactation (L1) and L10. Results are shown as the means +- s.e.m. of independent biological replicates (p18 and L1, n = 3; L10, n = 4). Two-way ANOVA followed by Tukey's multiple comparisons test was used to evaluate the statistical significance of differences. The Cish and Krt18 genes served as controls. b ChIP-seq data for STAT5a binding, H3K4me3 marks and Pol II loading at the Csn1s2a - Csn1s2b locus during pregnancy (p18) and lactation (L1 and L10). The orange shades indicate candidate enhancers and the blue shades are promoter regions. The Cish locus served as control (Supplementary Fig. 1 ). DE, Csn1s2b distal enhancer; IE, intronic enhancer; P, promoter. c - d TF binding and activating histone marks at the Csn1s2a ( c ) and Csn1s2b ( d ) genes at L10. The black bars indicate DNA binding motifs for STAT5 (TTCnnnGAA), GR (RGXACAnnnTGTXCY) and NFIB (TGGCA/TGCCA). The orange and blue shades indicate putative enhancers and promoters, respectively.

Supportive validation

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Experimental details: Identification of putative regulators of motor neuron maturation. a Identification of motifs enriched during maturation using DeepAccess. Each dot represents one of 108 consensus transcription factor binding motifs. The expected pattern effect ( x -axis) is a score of how differentially predictive each motif is of accessibility between two timepoints. For example, NFI motifs are highly predictive of accessibility at E13.5, P4, and P21 as compared to E10.5. b Motif enrichment using HOMER. Each column shows the most highly enriched motifs in the given condition. The left panel shows the most highly enriched motifs in the top 10k regions at each age. The two right panels show the most highly enriched motifs in the top 10k differentially accessible peaks. c ATAC-seq reads at 10k most significant genomic regions that are newly accessible between E10.5 and E13.5, E13.5 and P4, and P4 and P21. Each panel spans +-1 kb from the center of the peak. d HOMER outputs of motifs enriched in top 10k newly accessible peaks between P4 and P21. The motif logos show the de novo motif (top) and the best-matched known transcription factor motif (bottom) along with p value and prevalence. e Secondary motifs found in NFI-motif-containing peaks using HOMER, p value generated by HOMER. f Expression values of select transcription factors that bind to enriched motifs. Expression of all transcription factor family members is shown in Supplementary Fig. 6d . Error bars show SEM; n = 3 biological RNA-seq r

Supportive validation

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Experimental details: 1 Immunohistochemistry for glucocorticoid receptor (GR) in normal epithelia of the digestive system. The specificity of immunoreactivity for nuclear GR is shown ( A - C ). Strong reactivity for GR is found in nuclei (arrows) of muscle cells (mu) and some lymphocytes (ly) ( A ), but is completely blocked by neutralizing peptide absorption ( B ). In contrast, cytoplasmic reactivity (arrowhead) in epithelia (ep) remains unchanged after peptide blocking ( B ) and is still present after omission of primary antibody ( C ). Nuclear GR is present in squamous epithelia (arrow) of the oesophagus ( D ) and in chief cells (arrow), but not in parietal cells (arrowhead) of the stomach ( E ). GR is completely undetected in colonic epithelia (arrowhead), but present in mesenchymal cells (arrow) ( F ). Nuclear immunoreactivity of GR (arrow) is strong in hepatocytes ( G ), mild in epithelia of the common bile duct ( H ), strong in pancreatic islet cells (arrow) and comparatively weak in ductal epithelia (arrowhead) ( J ).

Supportive validation

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Experimental details: 2 Expression of glucocorticoid receptor (GR) in carcinoma of the human digestive system. GR immunoreactivity in oesophageal squamous cell carcinoma (SCC) ( A - F ). Diffuse strong positivity for GR is found in SCC A . This reactivity is completely blocked by peptide absorption ( B ) and completely eliminated after omission of the primary antibody ( C ), confirming its specificity. The green, red and blue fluorescence indicates reactivity of cytokeratin, GR and 4'-6-diamidino-2-phenylindole (DAPI), respectively ( D - F ). The composite image shows co-localization of GR and DAPI (purple signal, white arrow) ( F ), indicating that GR is restricted to the nuclei of carcinoma cells. In contrast to SCC, GR is not detected in primary oesophageal adenocarcinoma (ca), but is found in adjacent non-neoplastic squamous epithelia (ep) ( G ). GR is not detected in epithelia (ep) or adenocarcinoma (ca) of the stomach ( H ), but is found in mesenchymal cells (arrow). Compared with the lack of reactivity in conventional colonic adenocarcinoma ( I ), GR is strongly expressed in basaloid carcinoma ( J ), but consistently not detected in adjacent colonic mucosal epithelia (ep). GR is intensely immunoreactive in hepatocellular carcinoma ( K ), but negative in adenocarcinoma (ca) of the ampulla of Vater ( L ). Representative immunopositive and negative cases of cholangiocarcinoma ( M , N ) and pancreatic adenocarcinoma ( O , P ) are shown. The strong reactivity of GR in stromal cells and

Supportive validation

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Experimental details: Figure 2. Viral validation of conditional glucocorticoid receptor (GR) knockdown rat. ( A ) AAV8-hSyn-Cre administration to the basolateral amygdala (BLA) in SD:nr3c1 fl/fl rats (panel A1- site of injection, 4X). Note the absence of GR (purple) in Cre + neurons (green) (panels A2-4, 40x, yellow triangles). ( B ) AAV8-hSyn-Cre administration to the BLA in SD:nr3c1 wt rats (panel B1- site of injection, 4X). Note the presence of GR (purple) in Cre + neurons (green) (panels B2-4, 40x, white triangles). ( C ) AAV9.CamKII.HI.eGFP-Cre.WPRE.SV40 (CaMKIIalpha Cre) injected into SD:nr3c1 f/f rats results in decreased GR (C4 yellow arrows) in cells infected with virus as shown by GFP labeling (C2 yellow arrows) that are also CaMKIIalpha positive (C3 yellow arrows). CaMKIIalpha cells not infected with GFP, show endogenous GR expression (C1-4 white arrows). ( D ) CaMKIIalpha Cre injected into SD:nr3c1 wt rats (panel C2-4 40x white arrows) shows endogenous GR staining (D4) in cells infected with virus shown in GFP (D2) and CaMKIIalpha positive (D3). ( E ) AAVrg-pmSyn1-EBFP-Cre administration to the BLA in SD:nr3c1 fl/fl rats (panel E-1 site of injection, 4X) and retrograde trafficking of the virus to cell somas in the prefrontal cortex (PFC) (panel E2-4). Note absence of GR expression (purple) in Cre + neurons (green) (panel E5-7, 20x, yellow triangles). ( F ) AAVrg-CAG-GFP administration to the BLA in SD:nr3c1 fl/fl rats (panel F1- site of injection, 4X) and retrograde trafficking of the