“…Ten minutes after the second transfection, the cells were cultured with rifampicin (5 Â 10 26 M), roscovitine (5 Â 10 26 M), or vehicle in the presence or absence of various inhibitors for an additional 24 hours unless otherwise stated. Total RNA was extracted using TRIzol reagent from Invitrogen (Carlsbad, CA), and cDNA synthesized from 75 ng of total RNA was subjected to a quantitative real-time PCR as described previously elsewhere (Sugatani et al, 2012) with a Stratagene Mx3000P Real-Time QPCR System (Agilent Technologies Japan, Tokyo, Japan) using SYBR Premix Ex Taq reagent (TaKaRa Bio, Otsu, Japan) for the intercalation reaction with SYBR Green I according to the manufacturer's specifications for UGT1A1 and CYP3A4, as described previously elsewhere (Sugatani et al, 2010), and the protein phosphatase 1 (PP1) catalytic subunit beta isozyme, PP2 catalytic subunit alpha isozyme, and CaMKIIa (TaKaRa Bio). The thermal cycle conditions used were as follows: hold ).…”