We generated N-Me DNA sequences by PCR from mouse genomic DNA using a N-Me biotinylated forward primer (5’- ACTTCTACTGTATGCAGAATG-3’) and an unmodified N-Me reverse primer (5’- GTAATAAAAGACCTCTCTTCC-3’). We assembled extended nucleosome arrays using Chromatin Assembly Kit (Active Motif), following the manufacturer’s protocol. We generated compacted nucleosome arrays by adding histone H1 (Active motif) to extended nucleosome arrays and incubating for one hour at 27C. We incubated extended and compacted nucleosome arrays with GATA3 (Origene) for two hours at room temperature in binding buffer containing 10mM Tris pH 7.5, 1mM β-mercaptoethanol, 40mM KCl, 5mM DTT, 250ug/mL BSA, 1% ficoll and 5% glycerol. We digested nucleosome arrays with 40U/mL of DNAse I (NEB) for one minute at room temperature. DNA was purified, run in an agarose gel and transferred into a nylon membrane for chemoluminiscence detection using Streptavidin-AP conjugate (Roche) and CDP-Star (Roche). DNA smear products were quantified by plot profile analysis using Fiji and normalized to the total DNA content.
Gata 3
Manufactured by OriGene
Sourced in United Kingdom
GATA-3 is a transcription factor that plays a key role in the development and differentiation of T cells. It is a commonly used marker for identifying T cells and their subsets.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Streptavidin ap conjugate, by Roche (1 mentions)
Chromatin assembly kit, by Active Motif (1 mentions)
Cdp star, by Roche (1 mentions)
Fitc conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Nf κb, by Thermo Fisher Scientific (1 mentions)
Cox 2, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 conjugated anti goat igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindol dapi, by Thermo Fisher Scientific (1 mentions)
3 protocols using gata 3
1
GATA3 Binding on Compacted Nucleosomes
2
Immunofluorescence Analysis of Immune Signaling
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In order to evaluate activation of specific proteins, such as T helper cell transcription factors (T-bet for Th1 cells and GATA-3 for Th2 cells) and NF-κB/COX-2 for inflammation pathways, immunofluorescence analyses were conducted on individuals of four groups: control, OVA treatment, OVA-induced DEX treatment, and OVA-induced 50 mg/kg KRGWE treatment. Before the antibody binding step, the same materials detailed in the immunohistochemical analyses were used, in addition to T-bet (Biorbyt, orb7075, Cambridge, UK), GATA-3 (OriGene, TA305795, Rockville, MD, USA), NF-κB (ThermoFisher Scientific), or COX-2 (Invitrogen, PA1-9032, Carlsbad, CA, USA), which were used as primary antibodies for incubation at room temperature for 1 h. All of them were incubated with FITC-conjugated anti-rabbit IgG for 2 h (#315-095-003; Jackson Immunoresearch, West Grove, PA, USA) or Alexa Fluor 555-conjugated anti-goat IgG (ThermoFisher Scientific), and cells were counterstained using 4′,6-diamidino-2-phenylindole (DAPI; ThermoFisher Scientific).
Images were produced using a K1-Fluo confocal microscope (Nanoscope System, Daejeon, South Korea), and the fluorescence intensity was measured.
Images were produced using a K1-Fluo confocal microscope (Nanoscope System, Daejeon, South Korea), and the fluorescence intensity was measured.
3
Immunofluorescent Analysis of Th1 and Th2 Transcription Factors
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To confirm the intracellular distribution of Th1 cell transcription factor, T-bet, and Th2 cell transcription factor, GATA-3 immunofluorescent analysis was conducted for four groups: control, OVA, dexamethasone with OVA induction, and 1500 mg/kg socheongryongtang with OVA induction. T-bet (Biorbyt, orb7075, Cambridge, UK) or GATA-3 (OriGene, TA305795, Rockville, MD, USA) was used as a primary antibody. All samples were then incubated with fluorescein isothiocyanate (FITC)-conjugated IgG (Jackson Immunoresearch, 315-095-003, West Grove, PA, USA) or AlexaFlur 555-conjugated IgG (ThermoFisher Scientific, A-21127, Waltham, MA, USA) for 2 h. In order to counterstain, 4 ,6-diamidino-2-phenylindol (DAPI) was used (ThermoFisher Scientific, 62249, Waltham, MA, USA). The results were obtained by a K1-Fluo confocal microscope (Nanoscope System, Daejeon, Korea).
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