Total gsk3β

The primary antibodies used were Bcl-2, Beclin-1, PPARγ, p-AKT, total-AKT, p-GSK3β, total- GSK3β (Cell Signaling, USA); and β-actin (BD Biosciences Pharmingen, USA). The secondary antibodies used were anti-rabbit IRdye800, anti-mouse IRdye700 (1:50,000 dilution). SB216763 (3µM), Rosiglitazone (6mg/kg body weight) were purchased from Sigma-Aldrich, USA. Phosphatidylinositol 3-kinase (PI3K)/AKT inhibitor LY-294002 (10 µM) was purchased from Calbiochem, USA. The doses of the inhibitors and agonist used in this study were based on publications in the literature [28 (link)]. n-3 fish oil-based emulsion (10 g of TG/100 mL) was commercially prepared intravenous phospholipid-stabilized emulsions, and contained high concentrations of n-3 TG as previously described [27 (link)], [29 (link)]. n-3 TG emulsions were rich in EPA and DHA (~48% of total FA by weight) [27 (link)].

Protein abundance of protein kinase C epsilon (PKCε), glycogen synthase kinase-3β (GSK3β), and phospho-GSK3β in the left ventricles of the heart was measured with western blot analysis as described previously [15 (link), 28 (link)]. In brief, tissues were homogenized in a lysis buffer containing 150 mM NaCl, 50 mM Tris HCl, 10 mM EDTA, 0.1% Tween 20, 0.1% β-mercapto-ethanol, 0.1 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml aprotinin, pH 7.4. Homogenates were then centrifuged at 4°C for 10 min at 10,000g, and supernatants were collected. Samples with equal proteins were loaded on to the 7.5% polyacrylamide gel with 0.1% SDS and were separated by electrophoresis at 100 V for 90 minutes. Proteins were then transferred to the nitrocellulose membrane and incubated with primary antibodies for PKCε (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), phospho-GSK3β (Ser 9), and total GSK3β (Cell Signaling, Danvers, MA), respectively. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Chalfont St. Giles, Buckingghamshire, UK). Proteins were visualized with enhanced chemiluminescence reagents, and blots were exposed to Hyperfilm (GE Healthcare). Results were quantified with electrophoresis documentation and analysis. Band intensities were normalized to actin.

LiCl (GSK3β inhibitor), MG132 (Proteasome inhibitor), PD98059 (ERK1/2 inhibitor), SB230580 (p38 inhibitor), leptomycin B (LMB, Nuclear export inhibitor), zinc protoporphyrin IX (ZnPP, HO-1 inhibitor), 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-Fluorouracil (5-FU) and oxaliplatin were purchased in Sigma Aldrich (St. Louis, MO, USA). Antibodies against cyclin D1, phospho-cyclin D1 (Thr286), HA-tag, p-GSK3β, total-GSK3β, p-p38, total-p38, HO-1, Nrf2, cleaved PARP, TBP and β-actin were purchased in Cell Signaling (Bervely, MA, USA).

Protein was extracted from liver tissue with RIPA buffer with phosphatase inhibitors and protease inhibitors. Proteins in sodium dodecyl sulfate (SDS)-loading buffer were subjected to SDS-12 % polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. Monoclonal rabbit antibodies against p-GSK3β, total GSK3β, p-JNK, total JNK, Cyt c, caspase-3, cleaved caspase 3 and β-actin (Cell Signaling Technology, Santa Cruz, CA) were used at 4 °C overnight. Then the membrane was treated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The relative quantities of proteins were determined by a densitometer and expressed as absorbance units (AU).

Liver and eWAT (pooled samples from the same genotype and treatment, n = 2–4) were homogenized in lysis buffer containing 1% NP-40, 1% Triton-X 100, 1% SDS, 5 mM EDTA (pH 8.0), 50 mM Tris– HCl (pH 7.4), and protease inhibitor (P8340, Sigma, St. Louis, MO, USA) and phosphatase inhibitor cocktails (P5726 and P0044, Sigma, St. Louis, MO, USA). Protein concentrations were determined by BCA kit (Pierce, Thermo Scientific™, Waltham, MA, USA). Then, 10–20 µg tissue lysates were separated by 8% gel in SDS-PAGE and transferred to nitrocellulose membranes (Trans-Blot, Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% nonfat dried milk or 3% BSA (for phosphorylated proteins) for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C in 5% nonfat dried milk or 3% BSA (for phosphorylated proteins). Primary antibodies were purchased from Cell Signaling Technology, phospho-AKT (Ser473) (#9271), phospho-GSK3β (#8466), total AKT (pan) (#4691), and total GSK3β (#9315). Membranes were washed with TBS containing 0.1% (vol/vol) Tween 20, and incubated with 1:10,000 dilution of goat anti-rabbit horseradish peroxidase antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. Blots were visualized with enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA). The band densities were quantified using Image J software (NIH).

Cells were collected from the 60-mm culture dishes, washed with PBS, and lysed on ice in 100 μL of 1x RIPA buffer (Cell Signaling, Cat. 9806S) with 1x proteinase inhibitor cocktail (Roche, 11836170001) per dish. Protein concentrations were determined using a BCA Protein Assay kit (Pierce, Cat. 23228). Equal aliquots of protein (20 μg/lane) were run in SDS-PAGE and transferred by semi-dry transfer onto PVDF membranes. Then, membranes were blocked with 5% milk for 1 h and incubated with primary antibodies at 4°C overnight. After secondary antibody conjugation, the membranes were visualized by KwikQuant Imager using the ECL Western blotting detection kit (Biosciences, Cat. R1100). The primary antibody preparations against Mre11 (Cat.4895), Brca1 (Cat.9025S), total β-catenin (Cat. 8480), active β-catenin (Cat. 8814), pGSK3β (Ser9) (Cat.5558T), histone H3 (Cat. 4499s), and total GSK3β (Cat. 9315S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rad51 (Cat. sc-7410), Chk1 (Cat. sc-7898), and Per2 (Cat. sc-25363) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Chk2 (Cat. ab3292-500), CPT1A (Cat. ab128568), and NDUF12 (Cat. ab91521) were purchased from Abcam. β-actin is from Sigma (Cat. A5441).