For intracellular cytokine staining cultured cells were restimulated with PMA (50 ng/mL, Sigma Aldrich) and Ionomycin (1 μg/mL, Invitrogen, Life technologies) in the presence of Monensin (2.5 mM, Sigma Aldrich) for 5 h. To measure the cell viability (>80%) the cells were stained with Annexin-V FITC and 7-AAD according to the manufacturer's protocol (BD Bioscience). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin-containing buffer before washing and staining in different combinations with anti-CD4 (Beckman Coulter, Krefeld, Germany), anti-IL-17A, anti-IL-22, anti-IL-9, anti-IL-8, anti-AHR, anti-RORγt (ebioscience), and anti-IFN-γ (BD Bioscience). All dotplots and histograms shown refer to gated CD4+ T cells. Data were acquired on FACSCantoII (BD Bioscience) and were analyzed with FlowJo Software (Tree Star Inc., Ashland, OR, USA).
Anti ahr
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-AHR is a laboratory product designed to detect and quantify the Aryl Hydrocarbon Receptor (AhR) protein. It is a tool used in research applications to study the role of AhR in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 17a, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Monensin, by Merck Group (1 mentions)
Anti rorγt, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ, by BD (1 mentions)
Anti cd4, by Beckman Coulter (1 mentions)
Anti il 22, by Thermo Fisher Scientific (1 mentions)
Anti il 9, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
7 aad, by BD (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Human il 6, by Thermo Fisher Scientific (1 mentions)
Anti il17f, by Thermo Fisher Scientific (1 mentions)
Anti mtor, by Thermo Fisher Scientific (1 mentions)
Human il 1β, by Thermo Fisher Scientific (1 mentions)
Il 17f, by Thermo Fisher Scientific (1 mentions)
Mp4 25d2, by Thermo Fisher Scientific (1 mentions)
Human il 17a, by BioLegend (1 mentions)
8 protocols using anti ahr
1
Intracellular Cytokine Staining of T Cells
2
T Helper 17 Cell Differentiation
3
Nuclear and Cytosolic Protein Extraction
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Cytosolic and nuclear extracts were prepared as previously described [18 (link)]. Briefly, after 1 h ligand treatment, cells were washed and scraped into PBS. Cell pellets were resuspended in MENG (25 mM MOPS, 2 mM EDTA, 0.02% sodium azide, and 10% glycerol, pH 7.4) containing protease inhibitor cocktail (Roche) and homogenized with a stainless-steel Dura-Grind Dounce homogenizer (Wheaton Instruments, Millville, NJ, USA). Cell homogenates were centrifuged at 1000× g for 20 min, and the supernatant was then subjected to ultracentrifugation to generate cytosol. The nuclear pellet was washed three times with homogenization buffer, and then extracted with MENG containing 500 mM NaCl for 1 h, followed by centrifugation. Cell extracts were resolved on 8% tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred to PVDF membrane. Specific proteins were detected using anti-AHR (Thermo Fisher Scientific, Inc., Waltham, MA, USA), or anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibodies, and visualized with species appropriate biotin-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) and a subsequent incubation with 125I-streptavidin, which was generated as described [69 (link)].
4
Western Blot Analysis of AhR Signaling
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Protein extracts were run in 10% SDS-PAGE gels and transferred onto PVDF membranes by liquid transfer (Transfer buffer: 192 mM Glycine, 25 mM TrisBase, 20% methanol) at 200 mA during 90 minutes. Membranes were blocked overnight in TBST + 4% BSA (Sigma). Primary antibodies were incubated overnight at 4 °C: anti-AhR (1:500, mouse mAb, clone RTP1, ThermoFisher), anti-Lamin A/C (1:2000, mouse mAb, Cell Signaling), anti-GAPDH (1:4000, mouse mAb, Santa Cruz), anti-Lamp1 (1:2000, mouse mAb, H4A3 from the Developmental Studies Hybridoma Bank (DSHB), H4A3 was deposited to the DSHB by August, J.T./Hildreth J.E.K. (DSHB hybridoma product H4A3). Secondary mouse horseradish peroxidase-coupled antibody (DAKO) was successively incubated at room temperature for 2 h before detection with the Clarity Western ECL Substrate using the Chemidoc MP System (Bio-Rad). Quantifications were performed using the image Lab software (Bio-Rad). AhR nuclear protein levels were normalised to Lamin A/C protein levels. Lamp1 and GAPDH were used as purification controls for the cytoplasmic proteins.
5
Immunohistochemical Analysis of AHR, MT-1H, and Nrf2
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All explants were stained for the following: AHR (aryl hydrocarbon receptor), MT-1H (metallothionein) and Nrf2 (oxidative stress transcription factor). Each immunostaining was conducted on formol fixed paraffin-embedded sections with monoclonal antibody (anti-AHR, ThermoScientific, Waltham, MA, USA, MA1-514, clone RPT1; anti-MT1H Dako, M0639, clone E9; anti-Nrf2, abcam ab76026, clone EP1809Y), and biotin-conjugated secondary antibody. Staining was performed using the HRP–avidin/biotin complex (Vector Vectastain RTU Universal) and a violet substrate of peroxidase (VIP, Vector laboratories Inc., Newark, NJ, USA, SK4600). Immunostaining was performed using an automated slide processing system (Autostainer, Dako, Santa Clara, CA, USA) and assessed by microscopic observations.
6
Chromatin Immunoprecipitation of Transcription Factors
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Chromatin immunoprecipitation analysis was accomplished using the Magna ChIP™ A kit (Millipore). In brief, QKI5-silenced RAW 264.7 cells and control cells were incubated with medium or LPS (1 µg/ml) for 4 h, crosslinked in 1% formaldehyde for 10 min at room temperature and quenched with unreacted formaldehyde by the addition of 0.125 M glycine. Cells were washed twice with PBS and suspended in cell lysis buffer containing protease inhibitors, incubated on ice for 15 min, and centrifuged at 800 × g for 5 min. The pellet was homogenized in nuclei lysis buffer containing protease inhibitors and subjected to sonication on ice, followed by incubation overnight at 4°C with anti-Ahr (Thermo Fisher, clone RPT9), anti-p50 (Abcam), anti-p65 (Merck), anti-STAT1 (CST), normal mouse IgG, or normal rabbit IgG in the presence of protein A magnetic beads. The reactions were incubated overnight at 65°C to reverse the cross-links. DNA was purified by spin column and analyzed by PCR with the following primers: 5′-CGATGCTAAACGACGTCACATTGTGCA-3′ and 5′-CTCCAGAGCAGAATGAGCTACAGACAT-3′, specific for the κB site in the IL-6 promoter.
7
Quantitative Protein and RNA Analysis
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Pelleted cells were lysed in RIPA buffer (50 mM Tris pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.1% sodium deoxycholate; 1 mM PMSF; PhosSTOP and cOmplete PIC (Roche)) sonicated, and quantified by BCA assay. Equal sample amounts were then immunoblotted using Bolt gels and buffers (Thermo Fisher). Blots were blocked in 5% non-fat dry milk in TBSt (0.05% tween), washed in TBSt and incubated overnight at 4 °C with the following antibodies: anti-TDP-43 (ProteinTech; 12,892–1-AP; 10,782–2-AP); anti-Actin (Millipore; MAB1501); anti-α-synuclein (BD 610787); anti-ATXN2 (BD Biosciences; 611,378); anti-VCP (Thermo.; MA3–004); anti-AHR (Thermo.; MA1–514); anti-α-tubulin (Sigma-Aldrich; T5168). After washing, HRP-conjugated secondary antibodies (Jackson) were incubated with the blots the following day. Blots were activated with Pierce ECL chemiluminescent substrates (Thermo Fisher) and imaged using a ChemiDoc XRS+ Imager (BioRad). Band densitometries were assessed using Image Lab Software (BioRad).
RNA was collected from cultured cells by RNeasy minikit (Qiagen). cDNA was generated using High-Capacity cDNA Reverse Transcriptase (ABI). qPCR was performed using iQ SYBR green Supermix (Bio-Rad) on a 7900HT Fast Real-Time PCR system and the data was analyzed on SDS software. qPCR primer sequences are available in Additional file1 : Table S1.
RNA was collected from cultured cells by RNeasy minikit (Qiagen). cDNA was generated using High-Capacity cDNA Reverse Transcriptase (ABI). qPCR was performed using iQ SYBR green Supermix (Bio-Rad) on a 7900HT Fast Real-Time PCR system and the data was analyzed on SDS software. qPCR primer sequences are available in Additional file
8
Western Blot Analysis of Epigenetic Regulators
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Cell lysates (50 μg protein) were electrophoresed, transferred, and immunoblotted with specific antibodies. Anti-AhR, anti-TET1, and anti-TBP antibodies were purchased from Thermo Fisher Scientific, Inc.; the anti-DNMT1 antibody was from Abcam (Burlingame, CA, USA); the anti-DNMT3B antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the anti-p16INK4A and anti-EZH2 antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, USA), and the anti-MLL1 antibody was purchased from Active Motif (Carlsbad, CA, USA). The membranes with bound primary antibodies (1:1000) were reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies (Pierce, Rockland, IL, USA), and the protein bands were assessed by a western blotting detection kit (Amersham, Little Chalfont, Buckinghamshire, UK).
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