Anti phospho irs 1 tyr 632

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).

Differentiated C2C12 cells were harvested, and proteins were extracted with lysis buffer (PRO-PREP; Intron Biotechnology, Seoul, Republic of Korea) for 60 min at 4 °C. Protein samples (30 μg) were subjected to 12% SDS-PAGE, transferred to a nitrocellulose membrane (Amersham Bioscience, Westborough, MA, USA), and probed with the indicated primary antibody followed by a secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology). The signals were detected using enhanced chemoluminescence (ECL) kits (Amersham Bioscience). Anti-insulin receptor substrate (IRS)-1 (1:2500), anti-phospho-Akt (Ser473; 1:1000), anti-Akt (1:1000), anti-phospho-AMPK (Thr172; 1:1000), anti-AMPK (1:2500), anti-NFκBp65 (1:2500), anti-phospho-IκBα (Ser32; 1:1000), anti-phospho-eIF2α (Ser51; 1:1000), anti-eIF2α (1:1000), anti-PPARδ (1:2500), anti-PPARα (1:1000), anti-PPARγ (1:1000), anti-phospho-LKB1 (Ser428; 1:1000), anti-LKB1 (1:2500), anti-phospho-acetyl-CoA carboxylase (ACC) (Ser79; 1:1000), anti-ACC (1:2500), and anti-PGC1α (1:1000) antibodies were procured from Cell Signaling Technology (Beverly, MA, USA). Anti-phospho-IRS-1 (Tyr632; 1:1000) and anti-beta-actin (1:5000) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-METRNL antibody (1:1000) was supplied by Abcam (Cambridge, MA, USA).

Differentiated 3T3-L1 cells were harvested and proteins were extracted with lysis buffer (PRO-PREP; Intron Biotechnology, Seoul, Republic of Korea) for 60 min at 4°C. Protein samples (30 μg) were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences, Westborough, MA, USA) and probed with the indicated primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, USA). The signals were detected using enhanced chemiluminescence (ECL) kits (Amersham Biosciences). Anti-phospho Akt (Ser473; 1:1,000), anti-Akt (1:1,000), anti-phospho AMPK (Thr172; 1:1,000), anti-AMPK (1:2,500), anti-phospho NFκBp65 (Ser536; 1:1,000), anti-NFκBp65 (1:2,500), anti-phospho IκB (Ser32; 1:1,000) and anti-IκB (1:1,000) antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA). Anti-phospho IRS-1 (Tyr632; 1:1,000), anti-IRS-1 (1:1,000), anti-HO-1 (1:1,000) and anti-β-actin (1:5,000) were obtained from Santa Cruz Biotechnology.