Cells were seeded on glass coverslips and grown in DMEM, 5% charcoal stripped FBS for 3 days. After treatment with 100nM Dex or ethanol for 1 hour, cells were fixed with 4% paraformaldehyde for 10 min, permeabilize with 0.2% Triton X-100, PBS for 20 minutes, stained with anti-GR antibody (Santa Cruz, sc-8992), and visualized with anti-rabbit AF549 antibody (Invitrogen). Similar results were obtained using a second anti-GR antibody (Santa Cruz, sc-1003).
Anti gr antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-GR antibody is a laboratory reagent produced by Santa Cruz Biotechnology. It is designed to detect and bind to the glucocorticoid receptor (GR) protein, which is a nuclear receptor that mediates the effects of glucocorticoid hormones in cells. This antibody can be used in various research applications, such as western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of the GR protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit af549 antibody, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Dynamag 2 magnet, by Thermo Fisher Scientific (2 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Thermomixer, by Eppendorf (2 mentions)
Anti histone h3, by Santa Cruz Biotechnology (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Anti gapdh, by GeneTex (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Mini blot module, by Thermo Fisher Scientific (1 mentions)
Image studio version 5, by LI COR (1 mentions)
Immobilon fl transfer membrane, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Mifepristone, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
10 protocols using anti gr antibody
1
Glucocorticoid receptor immunofluorescence
2
Glucocorticoid receptor immunofluorescence
3
Glucocorticoid Receptor Phosphorylation Analysis
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Cells were washed with PBS and lysed in Trion X-100 lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% Trion X-100) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). Cell lysates were separated on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and transferred to PVDF membranes (Bio-Rad). Immunoblot signals were developed using SuperSignal West Pico Chemiluminescent Substrates (Pierce Biotechnology). Primary antibodies used in the study included anti-GR antibody (Santa Cruz), anti-GR(Phospho-Ser203) antibody (Assaybiotech), anti-GR(Phospho-Ser211) antibody (Cell signaling), anti-GR(Phospho-Ser226) antibody (Assaybiotech), anti-IKK2 antibody (Cell signaling), anti-histone H3 (Santa Cruz), anti-GAPDH (GeneTex), and anti β-actin antibody (Santa Cruz).
4
Immunoprecipitation of Glucocorticoid Receptor
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Cells were induced as specified in the figure legends and lysed in NP-40 lysis buffer. Immobilized (using 2mg/mL BSA) Dynabeads (50μL bead slurry, cat nr: 11204D, ThermoFisher Scientific) were added to the lysate and rotated for 1h at 4°C. Using the Dyna-Mag 2 magnet (ThermoFisher Scientific) the supernatant (precleared lysate) was separated from the beads and the protein concentration was measured via the Lowry method (see above). Then, 150μg precleared lysate was combined with 5μL anti-GR antibody (cat nr: sc-8992, Santa Cruz Biotechnology) and rotated for 1h at 4°C. Immobilized Dynabeads (50μL bead slurry) were added to the mixture and rotated for another 2h at 4°C. The bead-mixtures were washed three times with NP-40 lysis buffer and were denatured for 5’ at 95°C using a thermomixer (Eppendorf). The samples were subjected to WB analysis and anti-NCOA1 (SRC-1) antibody (cat nr: 128E7, Cell Signaling) was used to assay the interaction between immunoprecipitated GR and NCOA1.
5
Identification of ZBTB32 as a Glucocorticoid Receptor Interactor
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For the identification if ZBTB32 as a GR-binding protein, protein lysates were prepared from snap frozen adrenal glands (2 adrenals pooled from 1 mouse) by using RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche). Immobilized (using 2 mg/mL BSA) Dynabeads (50μL bead slurry, cat nr: 11204D, ThermoFisher Scientific) were added to the lysate and rotated for 1h at 4°C. Using the Dyna-Mag 2 magnet (ThermoFisher Scientific) the supernatant (precleared lysate) was separated from the beads and the protein concentration was measured via a Bradford protein assay (Bio-Rad). Then, 75 μg precleared lysate was combined with 5μL anti-GR antibody (cat nr: sc-8992, Santa Cruz Biotechnology) and rotated for 1h at 4°C. Immobilized Dynabeads (50μL bead slurry) were added to the mixture and rotated for another 2h at 4°C. The bead-mixtures were washed three times with protease inhibitor supplemented RIPA buffer and were denatured for 5′ at 95°C using a thermomixer (Eppendorf). The samples were subjected to WB analysis and anti-GR (1:1000, G5, sc-393232; Santa Cruz) and anti-ZBTB32 was used to assay the interaction between immunoprecipitated GR and ZBTB32.
6
GR Protein Expression Quantification
7
Molecular Mechanisms of Apoptosis Regulation
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Chemicals (Dex, DTX, and mifepristone) and the anti-β-actin antibody were purchased from Sigma (St. Louis, MO). Cpt was purchased from Qilu Pharmaceutical, Co. (San Dong, China). The anti-KLF5 antibody has been described previously [46 (link)]. The anti-GR antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-PARP, anti-caspase 3 and anti-caspase 7 antibodies were purchased from Cell Signaling Technology (Danvers, MA).
8
Dexamethasone Regulation of KLF5 Promoter
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Serum-starved HCC1937 cells were treated with 10 mM Dex or vehicle for 12 h before collection. The ChIP assay was performed following a protocol provided by Abcam (Cambridge, MA, USA). The diluted DNA-protein complex was incubated overnight with an equal amount of either anti-GR antibody or IgG control (Santa Cruz, CA, USA) at 4°C in the presence of protein A/G beads. After immunoprecipitation, chromosomal DNA was purified and analyzed using PCR to detect the predicted KLF5 promoter region. The primers used for amplifying the KLF5 promoter region surrounding the potential GRE site (-309 to -175) were 5’ -TACGTGCGCTCGCGGTTCTCT-3’ and 5’ -TCCGCTCTTCCACACGTA-3’.
9
Immune Cell Modulation Protocol
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CEP (HPLC purity: 99.1%, batch number: 0483686) was purchased from the Cayman Chemical Company. Methylprednisolone (MP) and verapamil (VP) were obtained from Sigma-Aldrich (St. Louis. Mo., USA). CEP, MP, and VP were dissolved in ethanol and stored at 4℃ until use. ConA was purchased from Seikagaku Kogyo Co. Tokyo, Japan. The WST-8 reagent was provided by Dojindo Molecular Technologies, Inc., Tokyo, Japan. The BD Cytometric Bead Array Human Th1/Th2/Th17 Cytokine Kit, FITC mouse anti-human CD4, APC mouse anti-human CD8 and CD25, and Alexa Fluor® 488 mouse anti-human Foxp3 antibodies were obtained from BD Biosciences (San Jose, CA, USA). The anti-P-glycoprotein antibody was purchased from Kamiya Biomedical Company, Seattle, USA (1 μg/mL, # MC-208). Anti-GR antibody was obtained from Santa Cruz Biotechnology, INC, Texas, USA (G-5, dilution:1:1000, # sc-393232). The anti-β-actin antibody was provided by Proteintech Group, Rosemont, USA (1:5000, # 66009-1-lg). The anti-TATA binding protein (TBP) antibody was purchased from Abcam (Cambridge, UK; dilution:1:1000, # ab818). RPMI-1640 and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY, USA). All other reagents used were of the highest quality available from commercial vendors.
10
Quantitative Immunofluorescence Analysis of GR Expression
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The A549 cells were grown on round coverslips. For the mouse studies, single-cell suspensions of fresh lung tissue specimens were prepared, and the cells were spun down onto cytospin slides. For the infant studies, slides of nasopharyngeal aspirates were made. The slides or coverslips were fixed with 4% paraformaldehyde. Blocking was performed using 5% bovine serum albumin. The slides or coverslips were then incubated with anti-GR antibody (1:50; Santa Cruz Biotechnology; USA) overnight at 4°C, followed by fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody (1:200; Beyotime, China) for 1 h at room temperature. The nuclei were stained with DAPI mounting medium for 30 min at room temperature. Immunofluorescent images were obtained using a fluorescence microscope (Olympus, Japan) and analyzed with Image-Pro Plus (Media Cybernetics, Bethesda, MD).
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