Six- to 8-week-old female C57Bl/6 wild-type mice were fed a diet deficient in methionine and choline for 5 weeks. Control animals received a diet supplemented with methionine (3 g/kg) and choline bitartrate (2 g/kg) (Dyets Inc., Bethlehem, PA). Poly(I:C) (InvivoGen, San Diego, CA), a synthetic dsRNA (5 mg/kg), or cytidine–phosphate–guanosine-rich DNA (CpG)-ODN (InvivoGen, San Diego, CA; 5 mg/kg) or LPS (Sigma-Aldrich Co., St. Louis, MO; 0.5 mg/kg) were injected intraperitoneally for 2 or 6 hours. The

study was approved by the Institutional Animal Use and Care Committee at the University of Massachusetts. Serum alanine aminotransferase (ALT) levels were determined NVP-LDE225 manufacturer using a kinetic method (D-TEK, Bensalem, PA), and liver triglyceride levels were assessed using

an L-Type Triglyceride H kit (Wako Chemicals USA Inc., Richmond, VA). Serum cytokine levels were determined by way of BD Cytometric Bead Array (BD Biosciences, Sparks, MD). Liver thiobarbituric acid reactive substances (TBARS) were assayed using whole liver homogenates and an Oxi-TEK TBARS assay kit (ZeptoMetrix Corp., Buffalo, NY). Serum high-mobility group box protein-1 (HMGB1) protein levels were measured by enzyme-linked immunosorbent assay (IBL Transatlantic, Toronto, Ontario, Canada). Sections of formalin-fixed livers were stained with hematoxylin and eosin. All slides were analyzed by way of microscopy. RNA was purified using an RNeasy kit (Qiagen Sciences, Germantown, MD) and on-column DNA digestion. selleck products Complementary DNA was transcribed with the Reverse Transcription System (Promega Corp., Madison, WI). Real-time quantitative polymerase chain reaction was performed using the iCycler (Bio-Rad Laboratories Inc., Hercules, CA) as described12; primer sequences

are shown in Table 1. Isolation of mitochondrial and cytosolic fraction from fresh liver tissue was based on the principle of differential centrifugation using a Mitochondrial Extraction kit (Imgenex Co., San Diego, CA). Whole liver lysates or mitochondrial oxyclozanide fractions were extracted and Western blotting was performed as described.12 The following antibodies were employed: MAVS (Santa Cruz Biotechnology Inc., sc-6881), cytochrome c (Imgenex, IMG101-A), caspase 1 p10 (Santa Cruz Biotechnology Inc., sc-514), cleaved caspase 8 (Imgenex, IMG5703), RIP3 (Abcam, ab72106), β-actin (Abcam, ab6276), β-tubulin (Abcam, ab6046), and Tim23 (BD Biosciences, 611222). A Native PAGE Novex Bis-Tris Gel System (Invitrogen Life Science, Carlsbad, CA) was used. Liver samples were lysed using 5% Digitonin as a mild detergent and separated on Native PAGE Novex 3-12% Bis-Tris gels. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane, fixed with 8% acetic acid, diluted in distilled water and identified with specific primary antibodies followed by horseradish peroxidase–labeled secondary antibodies and chemiluminescence assay.