Ahr h211

Cells were first washed with 1× PBS and resolved in RIPA buffer (100 mM Tris, 5 mM EDTA, 5% NP40; pH 8.0) with protease inhibitors (1 mM phenyl-methyl sulphonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin). Proteins were resolved by SDS-PAGE and then transferred to PVDF membranes. The blocking of non-specific binding was accomplished by adding 5% non-fat milk. After the application of primary antibody (AR (N-20), Santa Cruz, CA, USA; β-actin, Santa Cruz, CA, USA; AhR (H-211), Santa Cruz, CA, USA; BCRP/ABCG2 (BXP-21), Abcam, Cambridge, UK), secondary antibody (1:3000, HRP-goat-anti-mouse and HRP-goat-anti-rabbit) were applied for 1 h at room temperature. Signals were enhanced using an ECL chemiluminescence kit (Millipore, Danvers, MA, USA) and detected by ChemiDoc XRS+ (BioRad, Hercules, CA, USA).

Cells were fixed with fixative solution (3% paraformaldehyde and 2% sucrose solution) and permeabilized with 1% NP‐40. Cells were blocked with 5% bovine serum albumin (BSA) for 2 hours before incubation with Abs. AhR (H‐211, 1:50 dilution, Santa Cruz) and CYP2W1 (c‐7, 1:50 dilution, Santa Cruz) Abs were incubated at 4°C overnight. 2° Abs Alexa Fluor 488 goat anti‐Rabbit IgG (A11034, 1:1500, Invitrogen) and Alexa Fluor 488 goat anti‐Mouse IgG (A11001, 1:1500, Invitrogen). Samples were stained (DAPI; Invitrogen) for 20 minutes and mounted in the Antifade Mounting Medium, followed by analysis with a Nikon epifluorescence microscope.