Single-cell suspensions of the lamina propria of the small intestine were obtained, as described above. The cell surface was labeled with LinLD (catalog no. 423102; Biolegend, U.S.), CD45 (catalog no. A15395; ThermoFisher, U.S.), TCRβ,γδ (catalog no. MA5-28524; ThermoFisher, U.S.), NKp46 (catalog no. 250802; Biolegend, U.S.), CD127 (catalog no. PA5-79511; ThermoFisher, U.S.), RORγt (catalog no. ab104950; Abcam, Cambridge, UK), and GATA3 (catalog no. 386902; Abcam, Cambridge, UK). Cells were analyzed using LSRFortessa X-20 multi-dimensional high-definition flow cytometry (BD, Franklin Lakes, NY, USA), and the data were analyzed using FIowJo software version 10 (FlowJo, LLC, Ashland, OR, USA).
Gata3
Manufactured by Abcam
Sourced in United Kingdom, United States
GATA3 is a transcription factor that plays a critical role in the regulation of gene expression. It is involved in the development and differentiation of various cell types, including T cells, skin, and the urogenital system. GATA3 functions as a master regulator of T-helper 2 (Th2) cell differentiation and is essential for the expression of Th2-specific cytokines.
Automatically generated - may contain errors
Lab products found in correlation
Cebpb, by Abcam (6 mentions)
Prpf6, by Abcam (6 mentions)
Rorγt, by Abcam (3 mentions)
Ripa lysis, by Beyotime (3 mentions)
β actin, by Bioworld Technology (3 mentions)
N cadherin, by Elabscience (3 mentions)
Vimentin, by Elabscience (3 mentions)
β tubulin 3, by Immunoway (3 mentions)
E cadherin, by Elabscience (3 mentions)
Fluorescent labelled secondary antibody, by Proteintech (3 mentions)
E cadherin, by Abcam (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Rorγt, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
β actin antibody, by Abcam (1 mentions)
Vimentin antibody, by Abcam (1 mentions)
Twist1, by Abcam (1 mentions)
Snail, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
23 protocols using gata3
1
Immune Cell Profiling of Intestinal Lamina Propria
2
Cytokine and Transcription Factor Analysis
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The protein expression of IL-5, IL-13, OPN (R&D systems), GATA3 (Abcam), and RORα (Cloud clone) were determined by ELISA kits according to instructions. ECP level was detected by Unicap system.
3
GATA3 Regulation of Epithelial-Mesenchymal Transition
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U2OS and Saos2 cells were transfected with FLAG-GATA3 plasmid or GATA3 siRNA for 48 hours. Subsequently, cells were collected and lysis by RIPA protein extraction reagent supplemented with a protease inhibitor cocktail (Roche, Pleasanton, CA, USA). The concentration of protein was quantified using the BCA Protein Quantifcation kit (Pierce, Waltham, MA, USA) according to the manufacturer’s instructions. A total of 40 µg protein was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Sigma-Aldrich Co., St Louis, MO, USA). The membranes were blocked with 5% skim milk (OriGene Technologies, Inc., Beijing, People’s Republic of China) for 1 hour at room temperature. Subsequently, the membranes were incubated with antibodies, such as GATA3 (1:2,000, Abcam, Cambridge, UK), E-cadherin antibody (1:2,000, Abcam), N-cadherin antibody (1:2,000, Abcam), vimentin antibody (1:2,000, Abcam), slug (1:1,000, Abcam), snail (1:2,000, Abcam), twist1 (1:1,000, Abcam), and β-actin antibody (1:5,000, Abcam) overnight at 4°C. After washing with TBST buffer three times, the membranes were incubated with horseradish peroxidase-coupled (HRP) secondary antibody (1:5,000, Abcam) at room temperature for 1 hour. Finally, the blots were visualized using Western Lighting Chemiluminescence Reagent Plus (PerkinElmer, Inc., Waltham, MA, USA).
4
Protein Extraction and Western Blot Analysis
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Total protein was extracted via RIPA lysis (Beyotime, Shanghai, China) with phenyl-methane-sulfonyl fluoride and protease inhibitor. 10% SDS-PAGE gel electrophoresis with 30 μg protein per well was performed and then we transferred PVDF membranes. Antibodies PRPF6 (No. ab99292, 1:2000, Abcam, London, England), CEBPB (No. ab32358, 1:2000, Abcam, London, England), GATA3 (No. ab199428, 1:2000, Abcam, London, England), Vimentin (No. ab40497, 1:1000, Elabscience, Wuhan, China), E-cadherin (No. ab40285, 1:1000, Elabscience, Wuhan, China), N-cadherin (No. ab70061, 1:1000, Elabscience, Wuhan, China), β-tubulin III (No. YM0634, 1:2000, Immunoway, Wuhan, China), β-actin (No. AP0060, 1:5000, Bioworld, Wuhan, China) were incubated overnight at 4°C.
5
Chromatin Modulator Profiling Protocol
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Chaetocin (S8068), AMI-1 (S7884), BIX-01294 (S8006), GSK343 (S7164), 3-deazaneplanocin (S7120), and UNC0631 (S8071) were purchased from Selleek. Horseradish peroxidase (HRP) labeled secondary antibody (LK2003) and β-actin (KM9001T) were purchased from Sungene Biotech. Antibodies such as KMT1A (ab12405), H3K9me3 (ab176916), and GATA3 (ab199428) were purchased from Abcam.
6
Immunostaining and Confocal Imaging of Human Organoids
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The organoids were washed with cell recovery solution (Corning, USA) before being fixed for 30 min in 4% paraformaldehyde. After permeation and blocking, primary antibodies against human GATA3 (1,100, Abcam, UK), TFA2A (1,100, Abcam, UK), TFA2C (1,100, Abcam, UK), beta-hCG (1,500, Abcam, UK), E-cadherin (1,250, Abcam, UK), HLA-G (1,50, Abcam, UK), and PAPPA2 (1,100, Abcam, UK) were added and incubated at 4°C overnight. The following day, the organoids were sequentially incubated with fluorescence-labeled secondary rabbit or mouse antibody. Phalloidin and DAPI were added accordingly. Subsequently, the stained organoids were imaged using a ZEISS LSM 700 Confocal Laser Scanning Microscope and ZEN imaging software.
7
Immunofluorescence Staining of Organoids
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For imaging, organoids were cultured in 8-well chamber slides. Organoids were fixed by 4% formaldehyde (30 min, ambient temperature) and blocked (5% BSA, 0.2% Triton X-100, 0.1% Tween 20, in PBS; 1 h, ambient temperature), and incubated (1 h, 37 °C) with primary antibodies CK5 (BioLegend, Amsterdam, The Netherlands), CK 7 (Abcam, Cambridge, UK) and GATA3 (Abcam). Primary antibodies were incubated with complementary Alexa Fluor 488-labelled (Jackson ImmoResearch Europe, Cambridge, UK) secondary antibodies (1:250, 1 h, ambient temperature). Nuclei were counterstained by DAPI (DAKO), and the expression of the marker genes was visualized by microscopy (Zeiss Axiophot, Carl Zeiss AG, Oberkochen, Germany). Antibody diluent was 1% BSA in PBS. Samples without primary antibodies and samples stained with anti-rabbit IgG antibodies served as controls.
8
Plscr1 Expression in Lung Cell Populations
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To localize the expression of Plscr1 in various cell populations, lungs from WT mice with or without HDM challenges were sectioned, SPC (Santa Cruz, 13979), CX3CR1 (Thermo Fisher Scientific, 14-6093-81), or Gata-3 (Abcam, 199428) was labeled with red fluorescence (Alexa Fluor 594) and Plscr1 (Invitrogen, MA5-19636) was labeled with green fluorescence (Alexa Fluor 488). To localize the expression of Plscr1 and CRTH2, double-label immunofluorescence staining was undertaken using Paraffin-embedded lungs from WT and IL-13 Tg mice. Monoclonal anti-Plscr1 (Proteintech, 11582-1-AP), and anti-CRTH2 (Santa Cruz, sc-271898) were used in these evaluations. CRTH2 was labeled with red fluorescence (Alexa Fluor 594) and Plscr1 was labeled with green fluorescence (Alexa Fluor 488).
9
Western Blot Analysis of Cell Signaling Proteins
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At the end of the indicated treatments, the cells were rinsed with ice-cold PBC and then lysed with RIPA lysis buffer. Protein concentrations were determined using the BCA protein assay kit (Thermo Fisher Scientific). Proteins (50 µg) were run on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked using 5% fat-free milk for 1 h at room temperature. Antibodies used to probe the blots were Vimentin (ab24525), EpCAM (ab71916), PCBP1 (ab171681), E-Cadherin (ab76055), GATA3 (ab106625), cyclin D1 (ab16663), cyclin E (ab71535), CDK2 (ab232753), CDK4 (ab226474), CDK6 (ab84717) and TBP (ab28175) (all from Abcam). All antibodies were used at a 1:1,000 dilution. Blots were incubated with the primary antibodies overnight at 4°C. Blots were probed with TBP to confirm that equivalent amounts of proteins were loaded in each case. Post-incubation with primary antibodies, blots were washed thrice with 1X PBS and then incubated with goat anti-mouse IgG H&L (HRP) (ab6789; Abcam) or goat anti-rabbit IgG1 IgG H&L (HRP) (ab6721; Abcam) secondary antibody at 1:5,000 dilution for 1 h at room temperature. Post-incubation blots were washed thrice with 1X PBS. Blots were visualized using ECL Plus kit (Thermo Fisher Scientific, Inc.) and HyBlot CL film (Denville Scientific).
10
Immunoblotting Protocol for Protein Detection
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