Anti irs 1

3T3-L1 pre-adipocytes and eWAT samples were homogenised in commercial RIPA lysis and extraction buffer (cat. no. 89900, Thermo Scientific) and protein extraction and western blotting performed as described previously [15 (link)]. Each membrane was blocked in 5% non-fat milk powder added to 0.05% TBS/T (1 × TBS, 0.05% Tween 20) and further incubated overnight with the respective primary antibody (anti-IRS-1, 1:1000 dilution [cat no. 06-248 Upstate Biotechnology, Millipore, USA] and anti-Lunapark, 1:200 dilution [cat no. NBP1–80637, Novus Biologicals, USA]) solution. Following washing in TBS/T solution, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (1:10000) or anti-mouse (1:10000) antibody (Jackson Immuno Research, Stratech, UK). Antibody binding was detected using Super Signal West Pico Chemiluminescent substrate (Thermo Scientific) and an ImageQuant LAS 4000 machine and quantified using ImageQuant LAS 4000 software (GE Healthcare, UK). Coomassie Blue staining was used in all gels and anti-alpha-tubulin (cat no. 4074, Abcam, UK) antibody blotted on each membrane to confirm equal loading of proteins and equal transfer efficiency of samples.

Western Blotting was done as previously described [21 (link)]. Primary antibodies used were: anti-IRS-1 and anti-PI3K p85α subunit (Millipore, Lake Placid, NY, USA); anti-Akt1, anti-Akt2, anti-phospho-Akt Ser473, anti-phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2) Thr202/Tyr204, anti-phospho c-Jun N-terminal kinases 1 and 2 (JNK1/2) Thr183/Tyr185 and anti-phospho-p38 mitogen-activated protein kinase (MAPK) Thr180/Tyr182 (Cell Signaling, Danvers, MA, USA); anti-insulin receptor beta (IRβ), anti-PI3K p110β subunit, anti-protein kinase C ζ (PKCζ) and anti-suppressor of cytokine signalling 1 (SOCS1; Santa Cruz Biotechnology, Heidelberg, Germany); and anti-GLUT4 (Abcam, Cambridge, UK). Peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies were used (Jackson ImmunoResearch, Stratech, Newmarket, UK).

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.

Mice were anesthetized after 24 h of fasting, and 5 U of human insulin (Humalin R; Eli Lilly) was injected into the inferior vena cava. After 5 min, the liver and hind limb muscles were dissected and immediately frozen in liquid nitrogen. The extracted proteins were immunoprecipitated by anti-IRβ (Santa Cruz Biotechnology), IRS-1, and IRS-2 (Millipore) and immunoblotted by anti-IRS-1, IRS-2, and phosphotyrosine (4G10) (Millipore). For phospho-Akt (Ser-473) and Akt analysis, the extracted total proteins were immunoblotted with their respective antibodies (Cell Signaling Technology).

Western Blotting was done as previously described [21 (link)]. Primary antibodies used were: anti-IRS-1 and anti-PI3K p85α subunit (Millipore, Lake Placid, NY, USA); anti-Akt1, anti-Akt2, anti-phospho-Akt Ser473, anti-phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2) Thr202/Tyr204, anti-phospho c-Jun N-terminal kinases 1 and 2 (JNK1/2) Thr183/Tyr185 and anti-phospho-p38 mitogen-activated protein kinase (MAPK) Thr180/Tyr182 (Cell Signaling, Danvers, MA, USA); anti-insulin receptor beta (IRβ), anti-PI3K p110β subunit, anti-protein kinase C ζ (PKCζ) and anti-suppressor of cytokine signalling 1 (SOCS1; Santa Cruz Biotechnology, Heidelberg, Germany); and anti-GLUT4 (Abcam, Cambridge, UK). Peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies were used (Jackson ImmunoResearch, Stratech, Newmarket, UK).

The antibodies used were: anti-phospho JNK (Thr183/Tyr185) (#4668), anti-phospho STAT3 (Tyr705) (#9131), anti-STAT3 (#8719), anti-phospho p38 MAPK (Thr180/Tyr182) (#9211), anti-p38 MAPK (#9212), anti-phospho Foxo1 (#9461) and anti-Akt (#9272) from Cell Signaling Technology (MA, USA); anti-phospho IGFIR (Tyr1165/1166) (sc-101704), anti-JNK (sc-571), anti-phospho-Akt1/2/3 (Ser473) (sc-7985-R), anti-caspase 1 (sc-514), anti-Nrf2 (sc-722) and anti-Keap1 (sc-33569) from Santa Cruz (Palo Alto, CA); anti-phospho IRS1 (Tyr1179) (07-844), anti-phospho IRS1 (Ser 307) (07-247), anti-IRS1 (06-248), anti-p85α (06-195) and anti-HO1 (AB1284) antibodies from Merck Millipore (Merck KGaA, Darmstadt, Germany); anti-β-actin (A-5441) antibody from Sigma Chemical Co. (St Louis, MO); anti-Lamin B (aB16048) and FasL (aB68338) from Abcam (Abcam, Cambridge, UK). Anti-IGFIR antibody was a gift of S. Pons (CSIC, Spain).