Serum was stored at −80°C. Livers were

snap-frozen in liquid nitrogen for proteins or stored in RNAlater (Qiagen, Hilden, Germany) for RNA extraction, or fixed in 10% neutral-buffered formalin for histopathological analysis. BM was collected from the long bones of 8-week-old donor male mice by flushing with a 25g needle and passed through a cell strainer to remove clumps. Female mice were irradiated (9 Gy) from a cesium irradiator (Gammacell 40, Atomic Energy of Canada), and 4 hours later transplanted with 5 million freshly isolated donor BM cells by way of a single tail vein injection. Transplanted find more mice were housed in microisolator cages and placed on medicated water (Sulfamethoxazole/Trimethoprim, Hi-Tech Pharmacal) until engraftment was complete 6 weeks later, as discussed.14, 15 The engraftment rate for all transplanted mice was >90%, as indicated by polymerase chain reaction (PCR) analysis of peripheral blood 6 weeks posttransplant. Serum alanine aminotransferase (ALT) was determined using a kinetic https://www.selleckchem.com/products/Deforolimus.html method (D-Tek, Bensalem, PA). Liver triglyceride levels were assessed using the L-Type Triglyceride H kit (Wako Chemicals, VA). Mouse IL-1β enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D

Systems (Minneapolis, MN), mouse and human TNF-α, IL-1β and IL-10 kits from BD Bioscience (San Jose, CA) and mouse IFN-β kit from PBL Interferon Source (Piscataway, NJ). Whole-cell lysates were extracted from liver and equal amounts of proteins were separated on polyacrylamide gel and transferred to a nitrocellulose membrane. Target proteins were detected by western blot and immunostaining with specific primary antibody, followed by horseradish peroxidase-labeled

secondary antibody. Antibodies specific for IRF3 and IL-10 were from Santa Cruz Biotechnology (Santa Cruz, CA), antiphosphorylated IRF3 and IFN-β Janus kinase (JAK) antibodies were from Cell Signaling Technology (Danvers, MA) and from ProSci (Poway, CA), respectively. The specific immunoreactive bands of interest were detected by chemiluminescence (Amersham, Piscataway, NJ) and quantified by densitometric analysis. RNA was purified using the RNeasy kit (Qiagen Sciences, MD) and on-column DNA digestion. cDNA was transcribed with the Reverse Transcription System (Promega, Madison, WI). SybrGreen-based real-time quantitative PCR was performed using the iCycler (Bio-Rad Laboratories, Hercules, CA) as described13; primer sequences are shown in Table 1. Liver sections were stained with hematoxylin and eosin or oil-red-O and analyzed by microscopy as described.13 Anesthetized animals were perfused by way of portal vein with saline solution followed by digestion as described.13 The hepatocytes were separated by centrifugation, liver mononuclear cells (LMNCs) were purified by centrifugation in Percoll gradient.