Sc 53850

Quinine, 3-hydroxyquinine (3-OH quinine), and carbamazepine (internal standard [IS]) were purchased from Toronto Research Chemicals (Toronto, ON, Canada). Cells from the fall armyworm (Spodoptera frugiperda [Sf]21), Sf-900™ III SFM insect culture medium, fetal bovine serum, Cellfectin^® II Reagent, and the Bac-to-Bac™ baculovirus cell expression system were obtained from Invitrogen (Carlsbad, CA, USA). PrimeSTAR^® HS DNA polymerase, restriction enzymes, and a DNA ligation kit were purchased from TaKaRa Bio (Shiga, Japan). A human cDNA clone for oxidoreductase (OR) (catalog number SC100401) and a wild-type CYP3A4 cDNA clone (catalog number SC125488) were obtained from Origene (Rockville, MD, USA). Mouse monoclonal anti-OR antibody, anti-CYP3A4 antibody, and anti-CYP3 antibody were from Santa Cruz Biotechnology (sc-25270, sc-53850, and sc-365415; Santa Cruz, CA, USA). Rabbit monoclonal anti-CYP3A4 antibody was purchased from Abcam (ab 3572; Cambridge, UK). A regenerating system for the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) was obtained from Promega (Madison, WI, USA). High-performance liquid chromatography-grade organic solvents and liquid chromatography–mass spectrometry-grade acetonitrile were purchased from Merck (Darmstadt, Germany). All other chemicals and reagents used were of the highest purity available.

Cells were fixed with 4% paraformaldehyde in PBS for 10 min at 4 °C. After washing with PBS and treatment with 0.2% Triton X in PBS for 10 min, cells were pre-incubated with blocking buffer (10% goat serum in PBS) for 30 min at room temperature and then exposed to primary antibodies to albumin (diluted at 1/50, CLFAG2140, Cedarlane Laboratories, Burlington, Canada), α-fetoprotein (diluted at 1/100, MAB1368, R&D Systems, MN, USA), and CYP3A4 (diluted at 1/200, sc-53850, Santa Cruz Biotechnology, USA) in blocking buffer overnight at 4 °C. Following washing with 0.2% PBST, cells were incubated with secondary antibodies; either anti-rabbit or anti-mouse IgG conjugated with Alexa 488 or 546 (1:300) (Invitrogen) in blocking buffer for 30 min at room temperature. Then, the cells were counterstained with DAPI.