Thr172pampk

The myotubes were incubated for 20 min in DMEM-low glucose-Glutamax in the presence or absence of 100 nM insulin. Afterwards, the cells were harvested in a RIPA buffer (Sigma, St. Louis, MO, USA) complemented with 10 µL/mL protease inhibitor, 10 µL/mL phosphatase I inhibitor and 10 µL/mL phosphatase II inhibitor. Afterwards, the protein concentration was determined by BCA reagent (Thermo Scientific, Rockfold, IL, USA). 20 µg of total protein were run on 4–15% Mini-PROTEAN^® TGX™ Precast Gels (Bio-Rad, Hercules, CA, USA), electroblotted onto PVDF membranes (Millipore, Bradford, MA, USA), and immunodetected with ChemiDoc MP imaging system (BioRad, Hercules, CA, USA) while using the following primary antibodies: GLUT4 (Santa Cruz Biotech, CA, USA), Ser9 pGSK-3β, Thr172pAMPK, Ser473 pAkt (Cell Signaling Technology, Danvers, MA, USA), Tyr-989 pIRS-1 (Abcam, Cambridge, UK), and Thr642 pAS160 (Gene Tex, CA, USA). Histone H3 (Cell Signalling Technology, Danvers, MA, USA) served as an internal control, with the exception of pAkt, where the internal control was GAPDH (Cell Signalling Technology, Danvers, MA, USA). The bound antibodies were visualized by an ECL system (Thermo Fisher Scientific Inc., Rockford, IL, USA) and quantified using Chemi-Doc MP imaging system (Bio-Rad, Hercules, CA, USA).

Whole-cell lysates were made in ice-cold lysis buffer supplemented with phosphatase and protease inhibitors (50 mM Tris-Cl pH7.4, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mmoL/L Na₃VO₄, 20 mmoL/L NaF, 1 μg/mL aprotinin, 10 μg/mL pepstatin, 10 μg/mL leupeptin, 1 μM phenylmethylsulfonyl fluoride). Tissue lysates were obtained from flash-frozen tissue, which was lysed by bead beating in lysis buffer. Protein quantification was performed using the Bio-Rad protein assay kit and bovine serum albumin. 30 µg of protein was resolved in a 12% polyacrylamide gel in Tris-Glycine SDS buffer (Invitrogen), before being electrotransferred onto nitrocellulose membranes (Whatman, Dominique Dutscher). Membranes were blocked in 5% non-fat milk in 0.1% Tween 20 Tris-buffered saline for 1 h and probed with the primary antibodies. AMPK (RRID:AB_915794; dilution 1:1000), Thr¹⁷²P-AMPK RRID:AB_2169396; dilution 1:1000), AKT (RRID:AB_329827; dilution 1:1000), Ser⁴⁷³P-AKT RRID:AB_2315049; dilution 1:1000) and tubulin (RRID:AB_2288042; dilution 1:1000) antibodies were purchased from Cell Signaling Technologies. The gp130 antibody was obtained from Santa Cruz Biotechnology (RRID:AB_647629; 0.2 µg/mL).