Solution-phase amide HDX was carried out with a fully automated system as described previously [38 (link)] with slight modifications. The automation system (CTC HTS PAL, LEAP Technologies, Carrboro, NC) was housed inside a chromatography cabinet held at 4°C. PGC-1α 2-220 and rosiglitazone bound PPARγ was mixed at 1:1 molar ratio and incubated for 1.5h at 4°C for complex formation bef ore subjecting to HDX. For HDX reaction, 5 μl of 10 μM apo PGC-1α 2-220 or the complex (1:1 molar mixture of PGC-1α 2-220 and PPARγ) was diluted to 25 μl with D2O-containing HDX buffer (either pH 7.5 or pH 6) and incubated at 4 °C for 10s, 30s, 60s, 9 00s or 3,600s. Following on exchange, unwanted forward or back exchange was minimized and the protein was denatured by dilution to 50 μl with 0.1% (v/v) TFA in 3 M urea and 50 mM TCEP. Samples were then passed across an immobilized pepsin column (prepared in house; [39 ]) at 50 μl min-1 (0.1% v/v TFA, 15 °C); the resulting peptides were trapped on a C8 trap cartridge (Hypersil Gold, Thermo Fisher). Peptides were then gradient-eluted (4% (w/v) CH3CN to 40% (w/v) CH3CN, 0.3% (w/v) formic acid over 5 min, at 4 °C) acr oss a 1 mm × 50 mm C18 HPLC column (Hypersil Gold, Thermo Fisher) and subjected to electrospray ionization directly coupled to a high resolution (60,000) Orbitrap mass spectrometer (LTQ Orbitrap XL with ETD, Thermo Fisher). MS/MS data were acquired in separate experiments with a 60 minute gradient. Data-dependent MSMS was performed in the absence of exposure to deuterium and the amino acid sequence of each peptide used in the HDX peptide set were confirmed if they had a MASCOT score of 20 or greater and had no ambiguous hits using a decoy (reverse) database. For on-exchange experiments, the intensity weighted average m/z value (centroid) of each peptide’s isotopic envelope was calculated using software developed in-house [40 (link)]. For back exchange correction, full deuterium control was run as reported previously [41 (link)] and an average of 70% recovery (ranging from 60% to 80%) was estimated. To measure the difference in exchange rates between experiments, we calculated the average percentage deuterium uptake for native PGC-1α 220 following 10, 30, 60, 900 and 3,600 s of on exchange. From this value, we subtracted the average percent deuterium uptake measured for PGC-1α 220 bound with either LBDs of PPARγ, RORγ, or ERRγ.
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Nuclear Receptor Subfamily 1, Group F, Member 3
Nuclear Receptor Subfamily 1, Group F, Member 3
Nuclear Receptor Subfamily 1, Group F, Member 3 (NR1F3) is a transcription factor that plays a key role in regualting gene expression related to cellular differentiation, development, and homeostasis.
It is a member of the nuclear hormone receptor superfamily and binds to specific DNA sequences to activte or repress target genes.
NR1F3 is involved in a variety of biological processes, including lipid metabolism, circadian rhythm, and immune response.
Researching this important nuclear receptor can provide insights into its diverse functions and potential therapeutic applications.
It is a member of the nuclear hormone receptor superfamily and binds to specific DNA sequences to activte or repress target genes.
NR1F3 is involved in a variety of biological processes, including lipid metabolism, circadian rhythm, and immune response.
Researching this important nuclear receptor can provide insights into its diverse functions and potential therapeutic applications.
Most cited protocols related to «Nuclear Receptor Subfamily 1, Group F, Member 3»
Amides
Amino Acid Sequence
APO 10
ARID1A protein, human
Buffers
Chromatography
Deuterium
formic acid
Gold
High-Performance Liquid Chromatographies
Isotopes
Lewy Body Disease
Molar
Nuclear Receptor Subfamily 1, Group F, Member 3
Pepsin A
Peptides
PPAR gamma
PPARGC1A protein, human
Proteins
Rosiglitazone
Tandem Mass Spectrometry
Technique, Dilution
tris(2-carboxyethyl)phosphine
Urea
Actins
BCL6 protein, human
CXCR5 Receptors
DNA, Complementary
GATA3 protein, human
Gene Expression
IL6R protein, human
IL17A protein, human
Interferon Type II
Interleukin-17F
interleukin-21
Nuclear Receptor Subfamily 1, Group F, Member 3
Oligonucleotide Primers
Oligonucleotides
Real-Time Polymerase Chain Reaction
RNA-Directed DNA Polymerase
RORA protein, human
SYBR Green I
T-Lymphocyte
trizol
Activation Analysis
ATAC-Seq
B Cell-Specific Transcription Factor
CD4 Positive T Lymphocytes
CD8-Positive T-Lymphocytes
Cells
Chromatin Immunoprecipitation Sequencing
Gene Expression
Genes
Gene Silencing
Nuclear Receptor Subfamily 1, Group F, Member 3
T-Lymphocyte
cDNA Library
Cells
Heat-Shock Proteins 70
Insecta
Luciferases, Firefly
Luciferases, Renilla
Mammals
M Cells
Nuclear Receptor Subfamily 1, Group F, Member 3
Paragangliomas 4
Promega
Proteins
Immunohistochemistry, immunofluorescence (IF), and flow cytometry were used to define the phenotype of immunofibroblasts in the salivary glands of patients with pSS. Tissue was obtained from patients recruited in the OASIS cohort (Optimising assessments in Sjögren’s syndrome) at University of Birmingham under ethics no. 10-018.
IL13, IL4, and IL22 receptor expression was detected in both humans and mice, and differential biological effects were observed in fibroblasts stimulated with these molecules. To dissect these effects in vivo, we studied the dynamic response of a population of pdpn+ cells in a model of TLS assembly in wt mice and in mutants defective for IL13, IL4R, IL4, IL22, IL22R, LTβR, and Rorγ, as well as Rag2 mice. Mice were maintained in the Biomedical Service Unit at the University of Birmingham according to Home Office and local ethics committee regulations (University of Birmingham), under license no. P4B291FAA. IF, flow cytometry, and qRT PCR were used to assess differential effects in these mutants in the salivary glands of mice killed at different time points. Recombinant proteins for the molecules of interest and a blocking antibody against IL22 were used in gain- or loss-of-function experiments to dissect the requirements of these pathways in fibroblast maturation and TLS formation. Finally, to prove the effect that pdpn+ fibroblast deletion would exert on TLS, we used DM2 mice that express the diphtheria toxin receptor under the FAP promoter.
Detailed materials and methods can be found inSI Appendix .
IL13, IL4, and IL22 receptor expression was detected in both humans and mice, and differential biological effects were observed in fibroblasts stimulated with these molecules. To dissect these effects in vivo, we studied the dynamic response of a population of pdpn+ cells in a model of TLS assembly in wt mice and in mutants defective for IL13, IL4R, IL4, IL22, IL22R, LTβR, and Rorγ, as well as Rag2 mice. Mice were maintained in the Biomedical Service Unit at the University of Birmingham according to Home Office and local ethics committee regulations (University of Birmingham), under license no. P4B291FAA. IF, flow cytometry, and qRT PCR were used to assess differential effects in these mutants in the salivary glands of mice killed at different time points. Recombinant proteins for the molecules of interest and a blocking antibody against IL22 were used in gain- or loss-of-function experiments to dissect the requirements of these pathways in fibroblast maturation and TLS formation. Finally, to prove the effect that pdpn+ fibroblast deletion would exert on TLS, we used DM2 mice that express the diphtheria toxin receptor under the FAP promoter.
Detailed materials and methods can be found in
Antibodies, Blocking
Biopharmaceuticals
CREB3L1 protein, human
Deletion Mutation
Diphtheria Toxin Receptor
Fibroblasts
Flow Cytometry
Homo sapiens
IL4R protein, human
IL22 protein, human
Immunofluorescence
Immunohistochemistry
Interleukin-13
Mus
Nuclear Receptor Subfamily 1, Group F, Member 3
Patients
Phenotype
RAG2 protein, human
Recombinant Proteins
Regional Ethics Committees
Salivary Glands
Sjogren's Syndrome
Tissues
Most recents protocols related to «Nuclear Receptor Subfamily 1, Group F, Member 3»
Eight- to 12-wk-old C57BL6/J male wild-type mice (Charles River Laboratories) were used for experimental purpose. Mice were provided food and water ad libitum, under 12-h light (6 AM to 6 PM; ZT0-ZT12) and 12-h dark (6 PM to 6 AM; ZT12-ZT0) conditions. Hepatocyte-specific ablation of HP1α (HP1α hep−/−), HP1β (HP1βhep−/−), HP1γ (HP1γhep−/−), RevErbα (RevErbαhep−/−), RORα/RORγ (RORα/RORγhep−/−), and E4BP4 (E4BP4hep−/−) was generated by crossing “floxed” female mice with albumin-CreERT2 floxed male mice (25 (link)), and subsequent tamoxifen injections were given for 5 d. All floxed mice were generated and maintained in IGBMC/Institut Clinique de la Souris (ICS). Genotyping was performed by PCR on genomic DNA isolated from mouse tails. All experiments were performed under light–dark (L/D) conditions, with ZT0 being the start of the light period (6 AM) and ZT12 the start of the dark period (6 PM). Mice were sacrificed at 4 h interval starting at ZT0 and fed a “normal” laboratory chow diet. Breeding, maintenance, and experimental manipulations were approved by the Animal Care and Use Committee of IGBMC/ICS.
Albumins
Animals
Diet
Females
Food
Genome
Hypomenorrhea
Light
Males
Mice, House
NOS2A protein, human
Nuclear Receptor Subfamily 1, Group F, Member 3
Rivers
RORA protein, human
Tail
Tamoxifen
Mouse: C57BL/6J (Charles River laboratories), Mouse: Alb-CreERT2/E4BP4 hep−/− [Mouse Clinical Institute (ICS)], Mouse: Alb-CreERT2/BMAL1 hep−/− [Jackson laboratories B6.129S4 (Cg)-Arntltm1Weit/J], Mouse: Alb-CreERT2/RevErbα hep−/− [Mouse Clinical Institute (ICS)], and Mouse: Alb-CreERT2/RORα/RORγ hep−/− [Mouse Clinical Institute (ICS)].
Mice, House
Nuclear Receptor Subfamily 1, Group F, Member 3
Rivers
First, 8 to 12-wk-old C57BL6/J male wild-type (WT) mice were from Charles River Laboratories. The mice were provided food and water ad libitum, under 12-h light (6 AM to 6 PM; ZT0–ZT12) and 12-h dark (6 PM to 6 AM; ZT12–ZT0) conditions. Hepatocyte-specific ablation of Bmal1 (Bmal1hep−/−), RevErbα (RevErbα hep−/−), RORα/RORγ (RORα/RORγ hep−/−), and E4BP4 (E4BP4 hep−/−) was generated by crossing “floxed” female mice with albumin-CreERT2 "floxed” male mice (61 (link)), and subsequent tamoxifen injections were given for 5 d. Bmal1-floxed mice were obtained from Jackson Laboratories (B6.129S4 (Cg)-Arntltm1Weit/J), whereas all other floxed mice were generated and maintained in Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)/Institut Clinique de la Souris (ICS). Genotyping was performed by PCR on genomic DNA isolated from mouse tails. All experiments were performed under light–dark (L/D) conditions, with ZT0 being the start of the light period (6 AM) and ZT12 the start of the dark period (6 PM). Mice were killed at a 4-h interval starting at ZT0. All mice were fed a normal laboratory chow diet. Breeding, maintenance, and experimental manipulations were approved by the Animal care and Use Committee of IGBMC/ICS.
Albumins
Animals
Diet
Females
Food
Genome
Hypomenorrhea
Light
Males
Mice, House
NOS2A protein, human
Nuclear Receptor Subfamily 1, Group F, Member 3
Rivers
Tail
Tamoxifen
The cell suspension obtained in the previous section was preincubated with Mouse BD Block purified antimouse CD16/CD32 mAb (394,656; clone: 2.4G2; 1/100; BD Biosciences) for 10 min at 22°C. The following antibodies were used for the gating of the innate lymphoid cells. Cell suspensions were incubated with a mixture of Biotin-CD3e (100,304; clone: 145-2C11; 1/200; eBioscience, Inc.), Biotin-CD45R/B220 (103,204; clone: RA3–6B2; 1/200; eBioscience, Inc.), Biotin-Gr-1 (108,404; clone: RB6-8C5; 1/200; eBioscience), Biotin-CD11c (117,304; clone: N418; 1/200; eBioscience, Inc.), Biotin-CD11b (101,204; clone: M1/70; 1/200; eBioscience, Inc.), Biotin-Ter119 (116,204; clone: TER-119; 1/200; eBioscience, Inc.), Biotin-FceRIa (134,304; clone: MAR-1; 1/200; eBioscience, Inc.), Brilliant Violet 510-Streptavidin (405,233; 1/500; eBioscience, Inc.), PE-Cy7-CD127 (135,014; clone: A7R34; 1/100; eBioscience, Inc.), Pacific Blue-CD45 (103,116; clone: 30-F11; 1/100; eBioscience, Inc.), and Fixable Viability Dye eFluor 780 (1/400; eBioscience, Inc.) for 20 min at 4°C. The cell suspension was washed twice with 2% FBS/PBS and fixed with fixation buffer (420,801; BioLegend, Inc.) for 30 min. After washing with 2% FBS/PBS, the cell suspension was incubated with the mixture of PE-GATA-3 (clone TWAJ, 1/50; eBioscience, Inc.), (clone AFKJS-9, 1/50, eBioscience, Inc.), and FITC-T-bet (clone 4B10, 1/50; BioLegend, Inc.)60 (link),61 (link) (Figure S1). We used the following antibodies for gating of M1 and M2 macrophages: FITC-CD206 (MA516870; clone: MR5D3, 1/50, eBioscience, Inc.), PE-F4/80 (12,480,182; clone: BM8, 1/50, eBioscience, Inc.), APC- CD45.2 (17,045,482; clone: 104, 1/50; eBioscience, Inc.), PE-Cy7-CD11c (25,011,482; clone: N418, 1/50, eBioscience, Inc.), and APC-Cy7-CD11b (47,011,282; clone: M1/70, 1/50; eBioscience, Inc.)62 (link) (Figure S2). We analyzed the stained cells using flow cytometry Canto II, and the data were analyzed using FlowJo (version 10; TreeStar, Inc.) ( ).
Antibodies
Biotin
biotin 1
Buffers
Cells
Clone Cells
Flow Cytometry
Fluorescein-5-isothiocyanate
GATA3 protein, human
ITGAM protein, human
Lymphoid Cells
Macrophage
Mus
Nuclear Receptor Subfamily 1, Group F, Member 3
Streptavidin
Viola
The three protein domains used as receptor targets (examples of the ROR family of receptors) in this paper are 7NPC, 7NP5, and 7KXD, according to the PDB database infrastructure [5 (link)]. The choice was made according to the following requirements: they all belong to the ROR family, they have similar and good data resolution, and each structure has one main chain; these domain data were collected using X-ray crystallography, and the publication year was 2021 [5 (link),6 (link),33 (link),34 (link)].
All mathematical background equations, methods, and files, along with the additional figures, are presented in the supplementary data (seeSupplementary Materials ) along with the additional figures. For the convenience of the reader, the whole project procedure is visualized in Figure 3 . It reflects the proposed workflow involving a number of steps; in the following sections, we refer to the particular steps presented in the chart. The study starts with setting up the environment (see Stage 1, Figure 3 ).
All mathematical background equations, methods, and files, along with the additional figures, are presented in the supplementary data (see
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Crystallography, X-Ray
Nuclear Receptor Subfamily 1, Group F, Member 3
Protein Domain
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More about "Nuclear Receptor Subfamily 1, Group F, Member 3"
Nuclear Receptor Subfamily 1, Group F, Member 3 (NR1F3), also known as the circadian locomoter output cycles kaput (CLOCK) gene, is a crucial transcription factor that plays a pivotal role in regulating gene expression related to cellular differentiation, development, and homeostasis.
As a member of the nuclear hormone receptor superfamily, NR1F3 binds to specific DNA sequences to activate or repress target genes, thereby influencing a variety of biological processes, including lipid metabolism, circadian rhythm, and immune response.
Optimizing your NR1F3 research can be greatly enhanced by utilizing the comprehensive resources and AI-driven tools available through PubCompare.ai.
This platform allows you to easily locate protocols from literature, preprints, and patents, and use AI-powered comparisons to identify the best protocols and products for your specific research needs.
When investigating the functions and potential therapeutic applications of NR1F3, researchers may utilize various laboratory techniques and reagents, such as TRIzol reagent for RNA extraction, the RNeasy Mini Kit for RNA purification, Ionomycin and PMA for cell stimulation, Lipofectamine 2000 and Opti-MEM for transfection, the Cytofix/Cytoperm kit for intracellular staining, the FACSCalibur flow cytometer for cell analysis, and M-MLV reverse transcriptase for cDNA synthesis.
The Infinite 200 PRO multimode reader can also be employed for various assays and measurements.
By leveraging the comprehensive information and AI-powered tools available through PubCompare.ai, researchers can optimize their NR1F3 studies, enhance reproducibility, and gain valuable insights into the diverse functions and potential therapeutic applications of this important nuclear receptor.
As a member of the nuclear hormone receptor superfamily, NR1F3 binds to specific DNA sequences to activate or repress target genes, thereby influencing a variety of biological processes, including lipid metabolism, circadian rhythm, and immune response.
Optimizing your NR1F3 research can be greatly enhanced by utilizing the comprehensive resources and AI-driven tools available through PubCompare.ai.
This platform allows you to easily locate protocols from literature, preprints, and patents, and use AI-powered comparisons to identify the best protocols and products for your specific research needs.
When investigating the functions and potential therapeutic applications of NR1F3, researchers may utilize various laboratory techniques and reagents, such as TRIzol reagent for RNA extraction, the RNeasy Mini Kit for RNA purification, Ionomycin and PMA for cell stimulation, Lipofectamine 2000 and Opti-MEM for transfection, the Cytofix/Cytoperm kit for intracellular staining, the FACSCalibur flow cytometer for cell analysis, and M-MLV reverse transcriptase for cDNA synthesis.
The Infinite 200 PRO multimode reader can also be employed for various assays and measurements.
By leveraging the comprehensive information and AI-powered tools available through PubCompare.ai, researchers can optimize their NR1F3 studies, enhance reproducibility, and gain valuable insights into the diverse functions and potential therapeutic applications of this important nuclear receptor.