Dgat2

The proteins were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 3% BSA, the membranes were probed with primary antibodies (SREBP-1c, PPAR-α, GPAM, DGAT1, and DGAT2 (Abcam, UK); FAS, AMPK-α, and pAMPK-α (Cell Signaling Technology, Danvers, MA, CA, USA); β-actin from (Thermo Fisher Scientific, Waltham, MA, USA)) overnight at 4 °C. The membranes were washed and incubated with an anti-rabbit IgG-HRP (GeneTex, Irvine, CA, USA) for 2 h. Proteins were imaged using an advanced enhanced chemiluminescence (ECL) advanced kit (Thermo Fisher Scientific, CA, USA). Protein expression was observed using the FUSION Solo System (Vilber Lourmat, Marne-la-Vallée, France), and semi-quantified using ImageJ (NIH).

Equal amounts of proteins (40 μg) were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrophoretic transfer of proteins onto nitrocellulose membranes. Blots were probed with antibodies against PPAR-α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PPAR-γ (Santa Cruz Biotechnology), PPAR-δ (Affinity Bio Reagent, Golden, CO, USA), tumor necrosis factor- (TNF-) α (AbDSerotec, MorphoSys UK, Oxford, UK), interleukin- (IL-) 6 (Bender MedSystems, Vienna, Austria), PPAR-γ coactivator- (PGC-) 1α (Abcam, Cambridge, UK), 5′ adenosine monophosphate-activated protein kinase 2α (AMPK2α) (Cell Signaling, Beverly, MA, USA), phosphorylated acetyl coenzyme A carboxylase (pACC) (Millipore, St. Louis, MO, USA), diacylglycerol acyltransferase 1 (DGAT1) (Abcam), DGAT2 (Abcam), cluster of differentiation 36 (CD36) (Abcam), phosphorylated AMPK2α (pAMPK2α) (Millipore), Akt (Cell Signaling), phosphorylated Akt (pAkt) (Cell Signaling), and secondary antibodies conjugated with horseradish peroxidase (HRP; Leinco Technology, St. Louis, MO, USA). Bound antibodies were detected with an enhanced chemiluminescence detection system (Millipore) and analyzed with AlphaEaseFC software (Alpha Innotech, San Leandro, CA, USA). Targeted bands were normalized to cardiac glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Sigma-Aldrich) to confirm equal protein loading.

Western blotting was carried out as described previously (27 ). Both αTubulin and Ponceau were used as loading controls. All quantitative data were calculated using Ponceau control. αTubulin is presented as a control in representative images. The following primary antibodies used in this study were obtained from Cell Signaling Technology: PPAR-γ (CAT#: 2443), SCD1 (CAT#: 2438), FASN (CAT#: 3180S), ACC (CAT#: 3662), phospho-ACC Ser-79 (Cat#: 3661), ATGL (CAT#: 2439), phospho-HSL Ser-660 (CAT#: 4126), phospho-HSL Ser-563 (CAT#: 4139), phospho-HSL Ser-565 (CAT#: 4137), HSL (CAT#: 4107), AKT (CAT#: 9272), phospho-AKT Ser-473 (CAT#: 9271), and phospho-AKT Thr-308 (CAT#: 9275). The primary antibodies obtained from Abcam were DGAT2 (CAT#: 237613) and αTubulin (CAT#: ab7291). Other primary antibodies include SREBP1 (CAT#: 2215, Novus Biologicals) and PEPCK1 (CAT#: 10004943, Cayman Chemical). The secondary antibodies used were Goat Anti-Rabbit IgG (H + L)-HRP conjugate (CAT#: 1706515, Bio-Rad Laboratories), and Goat Anti-Mouse IgG (H + L)-HRP conjugate (CAT#: 1706516, Bio-Rad Laboratories).