Srebp1

The samples were lysed using radioimmunoprecipitation assay buffer containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were determined using bicinchoninic acid protein assay (G-Biosciences, Maryland Heights, MO, USA). Thirty µg of protein lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer onto polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). The membranes were blocked at room temperature for one hour using TBS-T (10 mM Tris, 150 mM NaCl, and 0.05% Tween 20, (pH 7.6)) containing 10% skimmed milk, then probed with 1:1000 primary antibodies, such as FAS (Abcam, Cambridge, UK), ACC (Cell signaling, Danvers, MA, USA), SREBP1 (Novus, Centennial, CO, USA), ACOX1, CPT2 (Affinity Biosciences, Pottstown, PA, USA), PPARα (GeneTex, Alton Pkwy Irvine, CA, USA), HMGCR (Abcam, Cambridge, UK) and actin (Abnova, Taipei, Taiwan) at 4 °C overnight. Blots were then washed with TBS-T and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, UK) at room temperature for one hour. Protein bands were visualized using a chemiluminescence horseradish peroxidase substrate (Millipore, Burlington, MA, USA), and the relative signal intensity was quantified using ImageJ software.

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).

Mouse tissues were homogenized with a glass grinder in ice-cold RIPA buffer with protease and phosphatase inhibitors (GenDepot, Barker, TX). Cleared lysates were diluted to 4 µg/µl and 15–30 µg of protein was resolved on a 7.5–10% SDS-PAGE. For immunoblotting, the following primary antibodies and dilutions were used: rabbit antibodies total Akt (1:1000, #4691, Cell Signaling, Danvers, MA), pPKCζ T410 (1:1000, # 2060, Cell Signaling), pPKCβII S660 (1:1000, #9371, Cell Signaling), FAS (1:1000, #3180, Cell Signaling), HK2 (1:1000, #2867, Cell Signaling), PTEN (1:1000, #9188, Cell Signaling), primary mouse antibodies pAkt S473 (1:1000, #4051, Cell Signaling), β-tubulin (1:5000, #05–661, Millipore), SREBP1 (1:1000, NB #600-582, Novus Biologicals, Littleton, CO), CD68 (1:800, #ab 125212, AbCam, Cambridge, MA), and primary sheep antibody PKCε (1:2000, AF5134, R&D Systems, Minneapolis, MN). The following secondary antibodies were used: rabbit IgG (1:2000, W4011, Promega, Madison, WI), mouse IgG (1:2000, W4021, Promega), and sheep IgG (1:1000, HAF016, R&D systems). The signal was captured using an ImageQuant LAS 500 imager and analyzed by ImageQuant TL software (GE Healthcare, Chicago, IL).