Anti as160

Media, sera and antibiotics for cell culture were from Lonza (Walkersville, MD, USA). Protein electrophoresis and western blot reagents were from Bio-Rad (Richmond, VA, USA) and electrochemiluminescence reagents from Pierce (Rockford, IL, USA). Insulin was from Eli Lilly (Florence, Italy). The antibodies used were: anti-GLP-1 receptor (kind gift of Drs. Wanda Dolci and Bernard Thorens [10 (link), 23 (link)], anti-Insulin receptor (IR), anti-p-tyrosine (Biosource, Camarillo, CA), anti-IRS-1 (Millipore, Billerica, MA, USA), anti-p85 subunit of PI3-kinase, anti-Akt, anti-p-Akt S473, anti-p-AMPK T172, anti-AMPK, anti-p-ACC S78/80, anti-ACC, anti-p-JNK T183/Y185, anti-JNK, anti-p-AS160T642, anti-AS160 (Cell Signaling Technology, Beverly, MA, USA), anti-ERK1/2, anti-p-ERK1/2 (Santa Cruz, CA, USA), anti-GLUT4 antibodies [24 (link)], and anti-αTubulin (Sigma, St Louis, MO, USA). 5-amino-4-imidazole carboxamide riboside (AICAR), Compound C, and LY294002 were from Calbiochem (La Jolla, CA). SiRNAs were from Riboxx (Radebeul, Germany). Methylglyoxal (MGO) was from SIGMA-Aldrich (St Louis, MO, USA). Exenatide and liraglutide were kind gifts from Amylin and Novo Nordisk, respectively.

DMEM, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from GIBCO. Krebs-Henseleit prebuffer was purchased from Sigma. Anti-phospho AMPKα and anti-AS160 (a 160-kDa substrate of the Akt Ser/Thr kinase) antibodies were purchased from Cell Signaling (S1 Fig). Horseradish peroxidase (HRP)-conjugated anti-goat IgG, anti-mouse IgG, and anti-rabbit IgG antibodies were purchased from Jackson ImmunoResearch Ltd. Nitrocellulose membranes and X-ray films for western blot were purchased from Pall Corporation, Intron, AGFA, respectively. Supersignal West Pico chemiluminescent substrate and West Femto chemiluminescent substrate were purchased from Pierce. SYBR Green master mix for real-time PCR was purchased from Applied Biosystems.

The antibodies used included anti-IKKβ (Millipore, Billerica, MA, USA), anti-GAPDH, anti-IRS-1, anti-phospho-IRS-1(s318), anti-Bcl-2, anti-AS160, anti-phospho-AS160 (s588), anti-Akt, anti-phospho-Akt (s473), anti-phospho-p65 (s536), anti-Na-K-ATPase, anti-mouse IgG HRP-linked and anti-rabbit IgG HRP-linked antibodies, (Cell Signaling Technology, Boston, MA, USA), anti-Glut4, anti-p65 (Santa Cruz), goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 594 donkey anti-goat IgG, and Alexa Fluor 488 donkey anti-goat IgG (Invitrogen, Foster City, CA, USA). Mem-PER Plus Membrane Protein Extraction Kit and NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) were also used.