Genesnap

Raw 264.7 cells and hepatic tissue were harvested and homogenized with PRO-PREPTM protein extraction solution (iNtRON, Seoul, Korea) containing protease inhibitors. The cell and tissue lysates were centrifuged at 12,000 rpm for 30 min at 4°C. Then the protein concentration was measured by using the Bradford assay. An equal amount of protein was loaded on 6%–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to PVDF membranes, and then blocked using 5% skim milk for 1 h. Afterward, the membranes were incubated overnight at 4°C with the following primary antibodies: fatty acid synthase (FAS), SREBP1c, CCAAT/enhancer-binding proteins (C/EBPα), TNF-α (Cell Signaling Technology, Inc., Danvers, MA, United States, 1:500), carnitine palmitoyltransferase I (CPT1), acetyl-CoA carboxylase (ACC), phosphorylated ACC (p-ACC), PPARα (Santa Cruz Biotechnology, Santa Cruz, CA, United States, 1:200), α-tubulin (Sigma Aldrich, St. Louis, MO, United States 1: 4,000), anti-IL-6 (IL-6), and anti-C reactive protein (CRP) (Abcam, Cambridge, United Kingdom). After overnight incubation, the membranes were washed and incubated with the secondary antibody for 1 h at 20–25°C. The protein bands were detected using a ChemiDoc XRS+ imaging system (Bio-Rad, CA, United States) and Syngene GeneSnap (Syngene, Cambridge, United Kingdom).

The kidneys were ground and lysed on ice for 30 min. The lysate was centrifuged to remove tissue debris at 1945× g at 4 °C for 10 min. Each supernatant was centrifuged again at 9078× g at 4 °C for 30 min. Then, the final supernatant was collected for cytosolic extract. The pellet was re-crushed in a hypertonic lysis buffer for 1 h, and then the lysate was centrifuged at 9078× g at 4 °C for 20 min and the supernatant was used for nuclear extract. The protein concentration was quantified according to a BCA protein assay (ThermoFisher Scientific, Grand Island, NY, USA).
Thirty μg of each protein sample were loaded into an SDS-PAGE and transferred to poly-vinylidine fluoride (PVDF) membranes (Millipore, Marlborough, MA, USA). We used 8~12% SDS-PAGE gel according to the molecular weight (MW) of target protein(s). After the transfer, the membrane was blocked in 1~3% bovine serum albumin (BSA) in a phosphate buffed saline −0.1% Tween 20 (PBS-T), the membrane was incubated at 4 °C with each primary antibody. To detect primary antibodies, respective horseradish peroxide (HRP)-conjugated secondary antibodies were given to membranes. Protein bands were visualized using a chemiluminescent detector (Syngene, Cambridge, UK). Levels of targeted proteins were calculated using Syngene GeneSnap (Syngene, Cambridge, UK).

Western blots were performed as described earlier [54] (link). The membranes were incubated with primary antibodies directed against RED or SMU1 (Santa Cruz), A/PR/8/34 virions [55] (link), NS1 (kindly provided by Daniel Marc, INRA-Tours, France), NS2 (kindly provided by Florence Baudin, EMBL-Heidelberg, Germany), M1 (clone GA2B, Pierce), M2 (clone 14C2, Pierce), GAPDH (Pierce), tubulin (Calbiochem), the HA tag (clone 16B12, Covance), the Gaussia luciferase (New England Biolabs), with peroxidase-conjugated Streptavidin (IBA) and peroxidase-conjugated secondary antibodies (GE Healthcare), and with the ECL 2 substrate (Pierce). The membranes were scanned in a G-Box (Syngene), the chemiluminescence was acquired and quantified with the GeneSnap and GeneTools softwares (SynGene), respectively.