Anti ahr antibody

MDA-MD-468shAhR and Cal51shAhR were treated for seven days with vehicle or 750 ng/mL doxycycline (Dox) in DMEM with 10% Tet-Approved FBS. After treatment, cells were collected by trypsinization, washed with 1× PBS (Gibco, Invitrogen), and lysed using Triton X-100 lysis buffer (50 mM Tris pH 8.0, 400 mM NaCl, 10% glycerol, 0.5% triton X-100, protease inhibitors, and benzonase). Total protein concentration was measured using the Bradford method (BioRad), and 20 μg of protein was resolved using SDS-PAGE on 8% polyacrylamide gels. Protein was transferred to a nitrocellulose membrane at 4°C for one hour at 0.35A. Membranes were blocked with 5% nonfat milk in PBS + 0.1% Tween for one hour at room temperature, then incubated with 1:10,000 anti-AhR antibody (Santa Cruz, sc-5579) or 1:10,000 anti-β-Actin (Sigma, A5316) overnight at 4°C. Membranes were incubated with 1:10,000 goat anti-rabbit HRP or anti-mouse HRP secondary antibody for one hour at room temperature. Enhanced chemiluminescence reagents (Thermo Scientific) were applied to the membranes prior to exposure to x-ray film (Kodak).

EOC cells in test tubes were carefully washed twice with pre-chilled PBS. Non-denaturing lysis buffer (20 mM Tris HCl pH 8, 137 mM NaCl, 1% Nonidet P-40 (NP-40), 2 mM EDTA) was then added to the tubes. The cells were scraped off and moved to clean 1.5-ml tubes. 20 µl protein A/G agarose was added to the tubes to eliminate non-specific binding proteins, and the tubes were then shaken using a horizontal shaker for 10 min at 4 °C. The tubes were then centrifuged at 1000 g at 4 °C for 5 min, and the supernatant was transferred to new tubes while the protein A/G-agarose beads were discarded. A total of 500 μg of cell lysate per IP was used to immunoprecipitate AhR using anti-AhR antibody (Santa Cruz, CA, USA) at 4 °C, with the tubes shaken overnight. Then protein A/G plus agarose beads (Santa Cruz, CA, USA) were added directly to the supernatant and the mixture was shaken for 2 h at 4 °C. Next, it was centrifuged at 1000 g at 4 °C for 5 min, and the supernatant was removed. The beads were then washed twice with ice-cold PBS. Finally, 50 µl RIPA buffer (0.1% SDS, 150 mM NaCl, 50 mM Tris pH 8.0) and 6× sampling dye were added. The proteins were detected by western blotting using anti-AR and anti-AhR (Santa Cruz, CA, USA) antibody. Rabbit IgG was used as the control.