Western blotting gels

All Western blotting gels, membranes and electrophoresis equipment were purchased from Bio-Rad Laboratories. Total protein (20–30 µg) from treated HPHs were separated on SDS/PAGE (10% gels) and electrophoretically transferred on to PVDF membranes. Subsequently, membranes were blocked for 1 h at room temperature (24 °C) with 5 % non-fat dried skimmed milk powder in 0.1% TBS-T (Tris-buffered saline containing 0.1% Tween 20) before the incubation of primary antibody diluted in blocking buffer. Mouse monoclonal antibodies against human CYP2B6 and CYP3A4 were provided by Dr Frank Gonzalez (Laboratory of Metabolism, National Cancer Institute/National Institute of Health, Bethesda, MD, U.S.A.) and were diluted to 1:1000 for overnight incubation at 4°C. Anti-β-actin antibody (1:1000 dilution; Santa Cruz Biotechnology) was included as an internal control. The membranes were then washed three times with 0.1% TBS-T buffer and incubated with the appropriate secondary HRP (horseradish peroxidase)-conjugated antibody diluted to 1:4000–5000 in 2% non-fat dried skimmed milk powder in 0.1 % TBS-T for 2 h at room temperature. The membranes were developed using the Pierce ECL detection kit (Thermo Scientific).