Glycine

Tris, glycine, SDS, dimethylsulfoxide (DMSO), acrylamide and bis-acrylamide were purchased from Euromedex. Ponceau S, p-coumaric acid, bromophenol blue, brilliant blue R250, MgSO₄, Ca(NO₃)₂, COSO₄, CuCl₂, ZnSO₄, Na₂M₀O₄, Na₂B₄O₇, FeSO₄, VOSO₄, vitamins, HEPES, hydrogen peroxide, Tween 20, luminol, Triton X-100, sucrose, acetic acid, dithiothreitol (DTT), RNase A, and propidium iodide (PI) were obtained from Sigma. KCl, H₂SO₄ and KH₂PO₄ were from Merck. NaCl was from Fischer scientific. Tryptone peptone and yeast extract were obtained from Difco (Becton Dickinson). Ammonium persulfate (APS), N,N,N’,N’-tetra methyl ethylenediamine (TEMED), bovine serum albumin and Bradford reagents were purchased from BioRad. Propan-2-ol, ethanol, sorbitol, HCl, MgCl₂, MnCl₂ and (NH₄)₂SO₄ were from Prolabo. Glycerol was obtained from Acros organique.

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.