Anti cyp2c9

THLE-2 cell lysates obtained from the monolayers of THLE-2 cells expressing human drug metabolizing enzymes in 6-well plates were subjected to electrophoresis on 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with 8% (w/v) skim milk in phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20 (PBS-T) for blocking, and then reacted with specific primary antibodies, such as anti-CYP2C9 (1:500 dilution; Thermo Scientific), anti-CYP3A4 (1:500 dilution; Thermo Scientific), anti-UGT1A4 (1:500 dilution; Sigma), anti-actin antibody (1:1000 dilution, Sigma) or anti-Nrf2 antibody (1:1000 dilution, Abcam). Secondary antibodies (1:2000 dilution; Invitrogen) conjugated with horseradish peroxidase (HRP) were used and immunoreactive proteins were detected with the enhanced chemiluminescence reagent (Pierce). For Nrf2 nuclear accumulation experiments, around 10⁷ cells of THLE-2 cells were lysed after treating with acetaminophen for 12 h. Following treatment of cells, cytosolic and nuclear fractions were prepared using the classical method^{44 (link)} and stored at −80 °C prior to analysis. The HEK 293 cells expressing Nrf2 were prepared after transfecting pcDNA-Nrf2 (Addgene) with lipofectamine (Invitrogen) and used as a positive control Nrf2 expression.