Estrogen-Mediated Regulation of Detoxification Enzymes

MCF-10A cells were treated with E₂ (1 μM) in the presence and absence of SERMs (DMA, FDMA, Ral; 1 μM). Protein expression of CYP1B1 and 1A1 was analyzed using western blot experiments as previously described (27 (link)). Anti-CYP450 1B1 (Sigma; AV51761), Anti-CYP450 1A1 (Santa Cruz; sc-20772) and anti-β- actin (Cell signaling; #4967) antibodies were used as primary antibodies. Detoxification enzymes were also analyzed using anti-SULT1 (Santa Cruz,CA; sc-32928), anti-SULT1E1 (Santa Cruz, CA; sc-376009), anti-SULT1A1 (Santa Cruz, CA; sc-130883), anti-GSTpi (Cell signaling; #3369), anti-NQO1 (Santa Cruz; sc-32793), and anti COMT (Santa Cruz, CA; sc-25844) as primary antibodies. Antibodies were diluted in blocking solution (5% non fat milk in TBS with 0.1% tween 20). Blots were incubated with primary antibody overnight at 4 °C and with secondary antibody for 1 h at room temperature. Blots were visualized using chemiluminescence substrate (Thermo scientific, Rockford, IL). Imaging and analysis was done using FluroChem software (Cell Biosciences, Santa Clara, CA). Each protein band density was normalized to the respective β- actin band density and was represented as the relative protein expression. Three independent experiments were performed and results were represented as average ± SD.