Losartan

VSMCs were isolated from both young and old FXBN rat aortae as previously described [34 (link)]. Cells were sub-cultured (passage 3-6) in modified DMEM with 10% fetal bovine serum (FBS). After serum removal for 24 hours, cells were treated with Ang II (Sigma) or Losartan (Toronto Research Chemicals, E96000) or human vasorin protein (Sino Biological Inc., Beijing, China) or vehicle in 2.5% FBS for 24 hours. Other treatments are reported in the figure legends. Cultured VSMCs were lysed for protein and RNA isolation for Western Blot and q-RT-PCR analysis.

TaKaRa MiniBEST Whole Blood Genomic DNA Extraction Kit, PrimeSTAR HS DNA polymerase, restriction enzymes, and T4 DNA ligase were purchased from Takara Bio Inc. (Kusatsu, Japan). Spodoptera frugiperda (Sf) 21 cells, fetal bovine serum, Sf-900™ III SFM insect culture medium, Cellfectin II Reagent, and the Bac-to-Bac™ Baculovirus Expression System were purchased from Invitrogen (Carlsbad, CA, United States). Tolbutamide, 4-hydroxy Tolbutamide, losartan, losartan carboxylic acid, warfarin, 7-hydroxy warfarin, diazepam and midazolam (internal standard [IS]) were purchased from Toronto Research Chemicals (Toronto, Canada). Rabbit polyclonal anti-CYP2C9 antibody and mouse monoclonal anti-OR antibody were obtained from Abcam (Cambridge, United Kingdom) and Santa Cruz Biotechnology (Santa Cruz, CA, United States), respectively. A SuperSignal West Pico Trial Kit was obtained from Thermo Scientific (Rockford, IL, United States). A regeneration system for the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) was obtained from Promega (Madison, WI, United States). 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.