Cells and tumor tissues were lysed with RIPA Lysis buffer (Beyotime, Shanghai, China), and protein concentrations were determined by the BCA kit (Beyotime, Shanghai, China). Each sample was separated by 8-12% SDS-PAGE, blocked with 5% BSA (Albumin from bovine serum) for 2 h at room temperature, and incubated with primary antibodies to β-tubulin, Sox2 (#14962, 1:100, CST, MA, USA; sc-365964, 1:100, Santa Cruz, TX, USA; ab97959, 1:1000, Abcam, Cambridge, UK), AhR (ab2769, 1:500, Abcam, Cambridge, UK), STAT3 (#12640, 1:1000, CST, MA, USA), Y-STAT3 (ab76315, 1:2000, Abcam, Cambridge, UK), S-STAT3 (ab86430, 1:250, Abcam, Cambridge, UK), PCNA (#2586, 1:2000, CST, MA, USA), IDO1 (#51851, 1:500, CST, MA, USA), p53 (#2524, 1:1000, CST, MA, USA), p27 (#3686, 1:1000, CST, MA, USA), and p21 (sc-397, 1:200, Santa Cruz, TX, USA) overnight at 4 oC. Primary antibodies were detected with a goat anti-rabbit IgG-HRP (sc-2004, 1:2000, Santa Cruz, TX, USA) or anti-mouse IgG-HRP (sc-2005, 1:2000, Santa Cruz, TX, USA). The blots were developed using Super Signal West Pico chemiluminescent substrate (Millipore, Billerica, MA, USA).
Ab2769
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab2769 is a primary antibody product offered by Abcam. It is a recombinant monoclonal antibody targeting a specific protein. The core function of this product is to detect and bind to the target protein for research and analytical purposes.
Automatically generated - may contain errors
Lab products found in correlation
Igg isotype control, by Cell Signaling Technology (2 mentions)
Chip it express chromatin immunoprecipitation kit, by Active Motif (2 mentions)
Ab97959, by Abcam (1 mentions)
Sc 365964, by Santa Cruz Biotechnology (1 mentions)
Sc 397, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Merck Group (1 mentions)
Ab86430, by Abcam (1 mentions)
Anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ab76315, by Abcam (1 mentions)
Sc 372, by Santa Cruz Biotechnology (1 mentions)
Sc 722, by Abcam (1 mentions)
Tcs sp5, by Leica (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Ab9354, by Merck Group (1 mentions)
Anti actin, by Cell Signaling Technology (1 mentions)
Stat1, by Cell Signaling Technology (1 mentions)
Phospho stat1, by Cell Signaling Technology (1 mentions)
10 protocols using ab2769
1
Western Blot Analysis of Protein Markers
2
ChIP-qPCR Analysis of AhR Binding to SLC1A5 and SLC7A5
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HCT116 cells treated with 10 μmol/L CAI for 48 h were evaluated in a ChIP assay. The ChIP assay was performed using the ChIP-IT Express Chromatin Immunoprecipitation Kit (Active Motif, CA, USA) according to the manufacturer’s protocols. The antibodies used included anti-AhR (Abcam, Ab2769) and an IgG isotype control (Cell Signaling Technology, 3900). The SLC1A5 primer sequences are 5′-GGCATGCACGTGTCATCCTC-3′ (sense) and 5′-GGCATGCACGTGTCATCCTC-3′ (antisense). The SLC7A5 primer sequences are 5′-CAGGTCATCCTCTCATCCCTC-3′ (sense) and 5′-GCAGCCTCTCGTCAAGCCT-3′ (antisense). All sequences were designed to give amplicons <200 bp. The purified DNA (immunoprecipitated sample) was amplified by qPCR. The fold-enrichment method was used to normalize the ChIP-qPCR data.
3
Immunofluorescence Assay for Nrf2, AhR, and p65 in Astrocytes
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For immunofluorescence assay, C6 cells (3.0 × 105/well), primary astrocytes and mixed glial cells (2.0 × 105/well) were seeded on coverslips in 12-well plate and treated for 1 h with IS (30 μM). In some experiments with C6 cells, DPI (10 μM) and NAC (2 mM) were added 1 h before IS. In other experiments, CH-223191 (1 μM), was added 1 h before IS to C6 cells. Then cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS. After blocking with BSA and PBS, cells were incubated with rabbit anti-Nrf2 antibody (Santa Cruz Biotechnologies; sc-722; used at diluition 1:250), with mouse anti-AhR antibody (Abcam; ab2769; used at diluition 1:250) or with rabbit anti-p65 antibody (Santa Cruz Biotechnologies; sc-372; used at diluition 1:250). The slides were then washed with PBS for three times and fluorescein-conjugated secondary antibody (Immuno Reagents; used at diluition 1:2000) was added for 1 h. DAPI was used for counterstaining of nuclei. Coverslips were finally mounted in mounting medium and fluorescence images were caught using the Laser Confocal Microscope (Leica TCS SP5) as previously reported (Del Regno et al., 2015 (link)).
4
AhR Binding to Glut4 Promoter
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KGN cells were plated at a density of 2 × 105 cells and cultured for 24 hours with each treatment condition. After collection of cells, ChIP-qPCR was performed by using ChIP-IT Express Chromatin Immunoprecipitation Kits (Active Motif, CA, USA) according to the manufacturer’s protocol. The antibodies used included anti-AhR (Abcam, Ab2769) and an IgG isotype control (Cell Signaling Technology, 3900). The Glut4 primer sequences were 5′-ATGACCTCTGGTCACCAAACTG-3′ (sense) and 5′-GATCAGTCAGAAGCATAGGTGAC-3′ (antisense). Real-time PCR amplification was carried out and amplification of target gene is shown as fold enrichment compared to that of irrelevant antibody controls. The results were from 3 independent experiments followed by normalization to input signals and showed as mean ± SEM.
5
Protein Extraction and Western Blot Analysis
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Cells were collected, lysed in M2 lysis buffer and sonicated and protein concentrations were determined by the BCA kit (Applygen Technologies Inc., China). Then, the protein was run on an SDS–PAGE gel and transferred to nitrocellulose. Nitrocellulose membranes were blocked in 5% bovine serum albumin (BSA) and probed with antibodies overnight: anti-actin (Cell Signaling, Cat No. 3700; 1:1,000) and TopBP1 (Sigma, PLA0030, 1:1,000, ST, USA); anti-caspase 3 (Cell Signaling, Cat No. 9662; 1:1,000), cleaved caspase 3 (Cell Signaling, Cat No. 9664; 1:1,000), caspase 7 (Cell Signaling, Cat No. 9492; 1:1,000), cleaved caspase 7 (Cell Signaling, Cat No. 9491; 1:1,000), p27 (Cell Signaling, Cat No. 3686; 1:2,000), phospho-STAT1(Cell Signaling, Cat No. 9167; 1:1,000) and STAT1 (Cell Signaling, Cat No. 9172; 1:1,000); anti-β3 tubulin (Millipore, ab9354; 1:2,000, MA, USA) and IDO1 (Millipore, MABF850; 1:1,000, MA, USA) and anti-AhR abcam, ab2769, 1:1,000) and GCN2 (Abcam, ab137543; 1:1,000). Secondary antibodies conjugated to horseradish peroxidase were followed by enhanced chemiluminescence (Thermo fisher, 1:5,000, MA, USA). Results were confirmed by at least three independent experiments.
6
AHR Binding Profiling by ChIP
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ChIP studies were performed using an AHR-specific antibody (ab2769; Abcam, Cambridge, MA) and the ChIP kit (ab500; Abcam) according to the manufacturer’s protocol. Cells were fixed and sonicated to produce fragments averaging 500 bp. Following immunoprecipitation with AHR-specific antibody or normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX), DNA was purified and amplified using the following primers: Cyp1b1 primer: 5’-GTTTGGCGCTGGGTTAC-3’ and 5’-AGGTCGGAGCTGACTCTCT-3’ [104 (link)], Sox2 primer: 5’-CTGTGAGAAGGGCGTGAGAG-3’ and 5’- AAACAGCCAGTGCAGGAGTT-3’. The relative DNA amount was calculated using the ΔΔCt method. AHR and IgG control pull-down signal were normalized to input signal.
7
Chromatin Immunoprecipitation Protocol
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Cells were fixed in 1% formaldehyde for 10 min at room temperature, and immunoprecipitation was performed with an anti-AhR antibody (ab2769, Abcam, Cambridge, UK) or control IgG antibody with the Pierce Agarose ChIP Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The resulting immunoprecipitates, which included DNA, were analyzed by real-time PCR using Fast SYBR Green Master Mix (Applied Biosystems). The primers used are listed in Table 2 . Human genomic DNA extracted from THP-1 macrophages was used as a positive control (Cp values were around 11). Amplification of target sequences was confirmed by the DNA sequence. The percentage input of each sample was calculated from the Ct values.
8
Immunofluorescence Staining of Lung Tissue
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Lungs were treated with paraformaldehyde (42 (link)) perfusion, and tissues were fixed for 3 days in PFA 4% and transferred in sucrose 20% for 2 weeks. Half of right lung was included in tissue-tek® O.C.T (4583, tissue-tek®) and 10 μm frozen tissue were sectioned by using a cryomicrotome (Leica) at −20°C. For immunofluorescence staining, slides were incubated 30 min in citrate buffer at 80°C. Slides were washed with PBS 1× after each step, followed by 45-min incubation in saturation medium (TBS 1× , 1% BSA, 10% SVF, 0.3% triton X100). The primary antibody, an anti-AhR (10 μg/mL, ab2769, Abcam), was incubated overnight at 4°C. After washing, slides were treated with pontamin (0.05%, Chicago sky Blue 6B, Sigma) for 15 min. After washing (TBS 1×), slides were incubated with a secondary antibody, a goat-anti-mouse (4 μg/mL, ab150113, Abcam) during 1 h at room temperature. After washing, slides were incubated with DAPI (1/5,000 dilution) for 10 min, washed, and mounted in mowiol® (Sigma). Lung sections were observed under a fluorescence microscope Leica (Leica®, CTR6000) at 400× magnification for microphotography.
9
Quantification of TMAO and Related Metabolites
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Trimethylamine hydrochloride, TMAO, NADPH, magnesium chloride, tris (hydroxymethyl) aminomethane (Trizma® base), Trizma® hydrochloride, n-octylamine, methimazole, L-arginine and formic acid ( ≥ 95%) were purchased from Sigma-Aldrich (St. Louis, MO). Deuterated internal standard (d9-trimethylamine N-oxide) was purchased from Cambridge Isotopes (Cambridge, MA, USA). Optima LC–MS grade water, acetonitrile, and methanol was purchased from Fisher Scientific (Pittsburgh, PA). Taqman® primers used for mRNA quantification were purchased from Applied Biosystems (Foster City, CA). All fluorescent antibodies were purchased from Abcam (Cambridge, MA) (Codes: ab2769, ab8226, ab22717, ab126790, ab195627, ab186693 and ab175774).
10
Comprehensive Protein Extraction and Western Blot Analysis
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Total protein was extracted using a cell lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS (Beyotime, Shanghai, China) supplemented with each protease and phosphatase inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany) per 10 ml solution. Western blot was performed as previously described 57 (link). Primary antibodies against IRF1 (sc-514544x) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against P62 (5114), p-mTOR (5536), t-mTOR (2983), p-p70S6K (9234), p-4EBP1 (2855), PINK1 (6946) and Parkin (4211) were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). The antibodies against DRP1 (ab56788), MFN1 (ab57602), MFN2 (ab50838), OPA1 (ab90857), FIS1 (ab71498), AhR (ab2769), COXIV (ab14744), PGC1α (ab54481) and TFAM (ab131607) were purchased from Abcam (Cambridge, MA, USA). LC3 antibody (L7543) was purchased from Sigma-Aldrich.
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