Quantitative reverse transcription polymerase chain reaction (PCR) was performed using the TaqMan-PCR method as previously described. We evaluated the expression of the following genes: Acc1 (Mm01304282_m1)

for acetyl-coenzyme A (CoA) carboxylase 1, Srebf1 (Mm00550338_m1) for sterol regulatory element binding transcription factor 1 (Srebp-1c), Scd1 (Mm00772290) for stearoyl-coenzyme A desaturase 1, Fasn (Mm00662319_m1) for fatty acid synthase, Srebp-2 (Mm01306289_m1) for sterol regulatory element binding factor 2, Cd36 for the fatty acid (FA) translocase (Mm00432403), Dgat2 for diacylglycerol acetyl-transferase 2 (Mm00499530), Bax for Bcl-2 Associated X protein (Mm00432448_m1), Bcl-2 for B-cell lymphoma/leukemia-2 (Mm00477631_m1), Cpt1a (Mm00550438_m1) for carnitine palmitoyltransferase

1A, Ppara (Mm00440939_m1) Trametinib purchase for PPAR-α, ApoB Vemurafenib chemical structure (Mm01545164_m1) for apolipoprotein B, Mttp (Mm00435015_m1) for MTP, Ccl2 (Mm00441242_m1) for chemokine (C-C motif) ligand 2 (MCP-1), Emr1 (Mm00802530_m1) for cell surface glycoprotein F4/80 (F4/80), Tnf (Mm00443258_m1) for tumor necrosis factor-α, Tgfb1 (Mm00441724_m1) for transforming growth factor-β1, Hmgcr (Mm01282499_m1) for HMG-CoA reductase, Nrlh3 (Mm00443451_m1) for nuclear receptor subfamily 1, group H, member 3 (LXRα) and Ldlr (Mm00440169_m1) for LDL receptor. All experiments were performed in duplicate and all gene expressions were normalized to Hprt1 expression (ABI ID: Mm00446968_m1). Cell and liver samples were collected, and proteins were separated electrophoretically by sodium dodecylsulfate polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. The blots were incubated with primary MTP antibody (sc-33116, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Skp2 (L70) antibody, Phospho-SREBP-1c Avelestat (AZD9668) (Ser372) antibody, CDC20 antibody (Cell Signaling Technology, Danvers, MA, USA), anti-SREBP-1 antibody (2A4) (Abcam) and a horseradish peroxidase (HRP)-conjugated secondary antibody; donkey antigoat immunoglobulin (Ig)G-HRP (sc-2020, 1:5000; Santa Cruz Biotechnology) or antirabbit IgG-HRP (1:1500;

Cell Signaling Technology) or goat polyclonal secondary antibody to mouse IgG-HRP (Abcam). Thereafter, the membranes were visualized and electrochemiluminescence signals were quantified as previously described.[8] Mice liver microsomes were prepared as previously described,[18] and the supernatants were analyzed using a Bio-Rad DC protein assay kit (Bio-Rad, Richmond, CA, USA) and MTP Activity Assay (Roar Biomedical, NY, USA). To detect ubiquitin-conjugated proteins,[19] cell and liver homogenates were prepared in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40 and 0.5% sodium deoxycholate, and incubated with protein A and anti-MTP antibodies (612022; BD Transduction Laboratories, Tokyo, Japan) overnight. The immunocomplexes were washed, resolved on sodium dodecylsulfate polyacrylamide gels, and blotted onto polyvinylidene difluoride membranes.