Short interfering RNA (siRNA) knockdown of ERα and AhR was performed as described previously (Madak-Erdogan and Katzenellenbogen, 2012 (link)). Briefly, cells were seeded in 6-well plates using phenol red-free media at a density of 2.5 × 105 cells/well and transfected the next day with 25 nM control siRNA or 25 nM ERα siRNA or AhR siRNA using the DharmaFECT1 Transfection Reagent (GE Healthcare, Cat# T-2001-03). After 48 h, the cells were treated with compounds for the times indicated. siGE-NOME ESR1 siRNA SMART pool (Cat# M-003401-04) and ON-TARGET plus human AHR(196) siRNA SMART pool (Cat# L-004990-00) were obtained from GE Healthcare. The efficacies of siRNA knockdown of ERα and AhR were verified by qPCR and Western blot. Total cell lysate protein was subjected to Western blot analysis with anti-ERα antibody F-10 (Santa Cruz, sc-8002) and anti-AhR antibody H-211 (Santa Cruz, sc-5579).
H 211
Manufactured by Santa Cruz Biotechnology
Sourced in United States
H-211 is a laboratory equipment designed for spectrophotometric analysis. It is used to measure the absorbance or transmittance of light through a sample, allowing for the quantification of various substances within the sample.
Automatically generated - may contain errors
Lab products found in correlation
Anti erα antibody f 10, by Santa Cruz Biotechnology (1 mentions)
Dharmafect 1 transfection reagent, by GE Healthcare (1 mentions)
Cyp1b1, by Santa Cruz Biotechnology (1 mentions)
D5s6h, by Cell Signaling Technology (1 mentions)
Nucleospin column, by Macherey-Nagel (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Glycine, by Euromedex (1 mentions)
4 protocols using h 211
1
siRNA Knockdown of ERα and AhR
2
Immunohistochemical Analysis of AhR, Cyp1B1, and CD4
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Immunohistochemical labeling was performed as previously described [5 (link)]. Immunohistochemical staining for AhR (rabbit polyclonal antibody clone H211, Santa Cruz, 1/50 dilution) was performed using Ventana Autostainer (USA). Sections of some tumors were also immunostained with antibodies directed against Cyp1B1 (Santa Cruz, dilution 1/200) and CD4 (clone Sp35, Roche Ventana, USA). The antigen-antibody complex was visualized using DAB as chromogen, as previously described [5 (link)]. AhR immunostaining was analyzed blindly in duplicate by two specialists including a certified pathologist [5 (link)].
3
AhR ChIP-seq Experiments in Cancer and Lymphoblastoid Cells
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AhR ChIP-sequencing data were retrieved from the GEO database: GEO GSE90550 and GEO GSE116632 (Neavin et al., 2019 (link); Yang et al., 2018 (link)).
GSE90550 reports AhR ChIP-sequencing experiments performed using an AhR antibody (Santa Cruz, H-211) in triplicate, starting from MCF7 breast cancer cells treated with 10 nM TCDD or vehicle for 45 min (Yang et al., 2018 (link)).
GSE116632 reports AhR ChIP-sequencing experiments performed using an AhR antibody (Cell Signalling Technology, D5S6H) in duplicate, starting from a lymphoblastoid cell line (GM17212) treated with 1 μM 3-methylcholanthrene or vehicle for 24 h (Neavin et al., 2019 (link)).
GSE90550 reports AhR ChIP-sequencing experiments performed using an AhR antibody (Santa Cruz, H-211) in triplicate, starting from MCF7 breast cancer cells treated with 10 nM TCDD or vehicle for 45 min (Yang et al., 2018 (link)).
GSE116632 reports AhR ChIP-sequencing experiments performed using an AhR antibody (Cell Signalling Technology, D5S6H) in duplicate, starting from a lymphoblastoid cell line (GM17212) treated with 1 μM 3-methylcholanthrene or vehicle for 24 h (Neavin et al., 2019 (link)).
4
Chromatin Immunoprecipitation of Aryl Hydrocarbon Receptor
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The MCF-7 and MDA-MB-231 cells were treated with DMSO (Cont), and 1 nM TCDD or 10 µM glyceollin I or II for 1 h, washed twice with PBS and cross-linked for 10 min with 1.5% formaldehyde (Sigma-Aldrich, St Louis, MO, USA). The cross-linking reaction was stopped with 0.1 M glycine (Euromedex, Souffelweyersheim, France) for 1 min, and the cells were washed twice with cold PBS, scraped into 500 µL of cold PBS, spun 2 min at 3000 rpm and maintained at −80 °C. Then, the cells were lysed with lysis buffer [10 mM EDTA, 50 mM Tris-HCl (pH 8.1), 0.5% Empigen BB, and 1% SDS] and supplemented with a protease inhibitor (Roche) immediately before use. The cell lysates containing the cross-linked chromatin complexes were sonicated and immunoprecipitated with AhR antibody H-211 (sc-5579, Santa Cruz, Santa Cruz, CA, USA). The DNA was purified on NucleoSpin columns (Macherey-Nagel, Duren, Germany) using NTB buffer. ChIP DNA was used for real-time PCR. The primers used to amplify the AhR binding regions of the genes of interest are listed in Table 1 .
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