The data were processed and analyzed by Genespring GX software (Agilent Technologies). Significance was determined as >10-fold to the wildtype control samples. Specific messenger RNA (mRNA) levels were determined by quantitative real-time polymerase chain reaction (qPCR). RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and qPCR performed using cDNA generated from 1 μg of total RNA with SuperScriptII Reverse Transcriptase (Invitrogen). Primers for qPCR were designed using the Primer Express software (Applied Biosystems, Foster City, CA). qPCR reactions were carried out using the SYBR Green PCRmaster

mix (SuperArray, Frederick, MD) and an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Quantitation was carried out using LY2157299 purchase the comparative cycle threshold (CT) method and results were normalized to mouse β-actin. Rat UCP2 adenovirus was obtained from the Gene Neratinib order Transfer Vector Core (University of Iowa).18 For in vivo infection

of recombinant adenoviruses, 6 to 8-week-old wildtype mice were intravenously injected in the tail vein with 1.2 × 1010 infection units, in a total volume of 400 μL, of recombinant adenoviruses expressing UCP2 or with an adenovirus expressing Cre recombinase used as a control. Two days later, mice were administered APAP and the mice killed after 6 hours or 24 hours. Liver whole cell or mitochondrial extracts were prepared and subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Membranes were incubated with antibodies against CYP2E1, total JNK, p-JNK (Cell Signaling Technologies, Danvers, MA), or UCP2 (Santa Cruz Biotechnology, Santa Cruz, CA). JNK kinase

上海皓元 assays were performed using the nonradioactive SAPK/JNK kinase assay kit (Cell Signaling Technologies) according to the manufacturer’s instructions. APAP serum metabolites (APAP, APAP-NAC, APAP-glucuronide, APAP-CYS) were monitored as described.19 For serum palmitoylcarnitine, deproteinated serum samples from wildtype mice were analyzed by use of an API2000 triplequadrupole mass spectrometer (Applied Biosystems). Debrisoquine was used as internal standard. Samples were injected into a high-performance liquid chromatography (HPLC) system (PerkinElmer, Waltham, MA) using a Luna C18 column (Phenomenex, Torrance, CA; 50 × 2.1 mm i.d.). The flow rate through the column at ambient temperature was 0.3 mL/min with a gradient (methanol:water:acetonitrile, containing 0.1% formic acid) from 5:60:35 to 5:5:90 in a 6-minute run. The column was equilibrated for 1.5 minutes before each injection. The mass spectrometer was operated in the turbo ion spray mode with positive ion detection; the turbo ion spray temperature was maintained at 350°C and a voltage of 4.5 kV was applied to the sprayer needle. Nitrogen was used as the turbo ion spray and nebulizing gas.