For primers sequences, see Supporting Table S1. Real-time quantitative PCR data represent relative changes in hepatic gene expression. Results are reported as relative differences in gene expression with GAPDH used as an internal control. Samples were homogenized in a lysis buffer (50 mM Tris·HCl, pH 7.4, containing 150 mM NaCl, BMS-777607 solubility dmso 25

mM EDTA, 5 mM EGTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, and 1 mM DTT) containing protease inhibitor cocktail (Calbiochem) with phosphatase inhibitor. Homogenates were centrifuged at 12,000g for 5 minutes at 4°C and mixed with 5× reducing electrophoresis sample buffer (50 mM Tris·HCl, pH 6.8, containing 10% glycerol, 2% sodium dodecyl sulfate [SDS], 1% β-mercaptoethanol, and 0.02% bromophenol blue) and heated for 5 minutes at 95°C. Samples containing 10-25 μg protein were then resolved by 10% SDS polyacrylamide gel electrophoresis and transferred overnight onto nitrocellulose membranes by electrophoresis. Antibodies against SAPK/JNK, p-SAPK/JNK (Thr183/Tyr185) (81E11), Bip, http://www.selleckchem.com/products/azd6738.html IKKβ, eIF2α, p-eIF2α (Ser51), C/EBP homologous transcription factor (CHOP) (L63F7), were obtained from Cell Signaling Technology (Danvers, MA), ATF-6 was obtained from (Pro-Sci, CA), GADD34 (C-19), p-c-Jun (KM-1), and c-Jun(H-7a) were obtained

from Santa Cruz Biotechnology (Santa Cruz, CA), and ICAM-1 was obtained from Protein Tech Group (Chicago, IL). β-Actin antibody (Sigma Diagnostics, St. Louis, MO) was used to confirm equal protein loading among samples. Nuclear proteins were isolated from fresh liver tissue as described23 using a Nuclear Extract kit from Active Motif (Carlsbad, CA) according to the manufacturer’s 上海皓元医药股份有限公司 protocol. The NF-κB and AP-1 DNA binding

activity assays were performed using Trans-AM enzyme-linked immunosorbent assay (ELISA)-based kits from Active Motif (Carlsbad, CA) according to the manufacturer’s protocol. Nuclear extracts from liver tissue were incubated in a 96-well plate coated with oligonucleotide containing NF-κB or AP-1 consensus binding site. Activated transcription factors from extracts specifically bound to the respective immobilized oligonucleotide were detected using the antibodies to NF-κB p65 and p50 in NF-κB assays or c-Jun in the Ap-1 assay followed by a secondary antibody conjugated to horseradish peroxidase in an ELISA-like assay. Hepatic SAM and SAH levels were measured by high-performance liquid chromatography (HPLC) using the method of Henkel et al.24 For pharmacologic JNK inhibition experiments, cohorts of 5-10 C57BLKS mice were started on either the MCD or control diet.