Analysis of Aryl Hydrocarbon Receptor and CYP1A1Protein Expression

Heart and lung tissue were homogenized either in lysis buffer (20mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% triton X-100, Complete Mini Protease Inhibitor Cocktail Tablets [Roche, Mannheim, Germany]) for microsomal protein analysis or in a modified lysis buffer (25 mM HEPES, pH 7.4, 20mM NaMoO₄, 5mM EGTA, 3 mM MgCl₂, 10% glycerol, 50 μM leupeptin, 20 μg/ml aprotonin) for whole protein analysis. Protein concentration was determined by Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, Illinois), and microsomal- and total protein were analyzed for CYP1A1 and aryl hydrocarbon receptor (AHR), respectively, by Western blot. Protein samples were denatured by heating at 95ºC in SDS loading buffer for 5 min, resolved by electrophoresis on a 10% SDS-polyacrylamide gel, and transferred to a PVDF membrane. Membranes were probed using goat polyclonal anti-CYP1A1 (1:500, Santa Cruz Biotech, Dallas, Texas) or anti-AHR (Novus Biologicals, Littleton, Colorado), followed by donkey anti-goat IgG-HRP secondary antibody (1:20 000, Santa Cruz Biotech). Detection was performed using ECL reagent (Western Lighting-ECL kit, Perkin Elmer) and imaged on a ProteinSimple FluorChem system (ProteinSimple, Santa Clara, California). Membranes were then stripped using a mild stripping buffer, probed using a rabbit polyclonal anti-GAPDH primary antibody in heart homogenates, anti-β-actin in lung homogenates and anti-cytochrome P450 reductase (CYPOR) in microsomes (1:500 dilution) and a goat anti-rabbit IgG-HRP secondary antibody (1:2000 dilution). Band intensity was quantified using AlphaView SA digital image software.