Anti c ebpα 2295

Insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (Dex), and Oil Red O were obtained from Sigma (St. Louis, MO, USA). 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose] (2-NBDG) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Anti-Akt (#9272; 1:500), anti-phospho-Akt (p-Akt; Thr308; #9275; 1:500), and anti-C/EBPα (#2295; 1:1000) polyclonal antibodies were from Cell Signaling (Danvers, MA, USA). Anti-GLUT4 (G4048; 1:1000) polyclonal and β-actin (AC-15; A1978; 1:3000) monoclonal antibodies were from Sigma. Anti-PPARγ (H-100; 1:1000), anti-fatty acid synthase (FAS; H-300; 1:1000), anti-IRS-1 (C-20; 1:500), and anti-phospho-IRS-1 (Tyr-632; 1:500) polyclonal antibodies and anti-mouse, anti-rabbit, and anti-goat IgG, horseradish peroxidase-linked secondary antibodies (1:1000) were from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

Western blot analysis was performed as described previously^{8 (link)}. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane (Millipore, Billerica, MA, USA), and incubated with specific primary antibodies [anti-SREBP-1c (577036, BD Biosciences, Franklin Lakes, NJ, USA), anti-FAS (3180, Cell Signaling, Danvers, MA, USA), anti-C/EBPα (2295, Cell Signaling), anti-PPARγ (2435, Cell Signaling), anti-PDK1 (KAP-PK112, Enzo Life Science, Farmingdale, NY, USA), anti-PDK2 (sc-100534, Santa Cruz, Dallas, TX, USA), anti-PDK3 (sc-365378, Santa Cruz) and anti-PDK4 (ab214938, Abcam), anti-p-PDHE1α Ser232 (AP1063, Calbiochem, San Diego, CA, USA), anti-p-PDHE1α Ser293 (AP1062, Calbiochem), anti-HIF1α (NB100-123, Novus Biologicals, Centennial, CO, USA), anti-HSP90 (4874, Cell Signaling), and anti-β-tubulin (G098, Applied Biological Materials, Richmond, BC, Canada)].