Protein expression of Mrp2, Bsep, and P-gp was measured as previously described [21 (link)]. Briefly, total protein was obtained by homogenizing 100 mg of liver samples with equal volume of a tissue lysis buffer. Protein samples (30–50 µg) were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 4–15% gradient gel (Bio-Rad). Separated proteins were transferred onto a Potran 0.45 μm nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween20 (TBST) (Biosesang, Seoul, Republic of Korea) for 1 h and incubated with primary antibody at 4 °C for 12 h. Primary antibodies were used as follows: anti-Mrp2 antibody (ab203397, dilution 1:1000, Abcam, San Francisco, CA, USA), anti-Bsep antibody (ab217532, dilution 1:1000, Abcam), anti-P-gp antibody (E1Y7S, dilution 1:1000, Cell signaling technology, Danvers, MA, USA) and beta-actin antibody (1:1000, Cell signaling technology). The membrane was rinsed twice with TBST at 25 °C and treated with horseradish peroxidase-labeled anti-rabbit IgG antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Protein bands were visualized using a Luminata Forte enhanced chemiluminescence system (Millipore, Burlington, MA, USA), and beta-actin served as the loading control.
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