Glucose transporter 1 glut 1

Approximately 30 mg heart tissues collected from LV were homogenized in lysis buffer (Beyotime, Nantong, China) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma Chemical Co., St Louis, MO) and centrifuged. Protein lysates from each sample were then loaded onto 12% SDS-polyacrylamide gels (40 μg/well). Following electrophoresis, separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Thermo Scientific, USA). The blotted membrane was then incubated with primary antibodies specific for AMPKα and phospho-AMPKα^Thr172 (Cell Signaling Technology, Inc., USA), glucose transporter-1 (GLUT-1) (Cell Signaling Technology, Inc., USA), glucose transporter-4 (GLUT-4) (Cell Signaling Technology, Inc., USA), PGC-1α (Abcam, UK), and β-actin (Kangcheng Biological Co., China) overnight at 4°C. After washes, blots were then incubated with horseradish peroxidase-conjugated secondary antibody (Abbkine, Inc., USA) for 2 h. Finally, immunocomplexes were visualized by ECL Plus (Thermo Scientific, USA) and analyzed with Image Lab Software (Bio-Rad Laboratories, Inc., USA). Quantification of relative protein expression was normalized to β-actin and expressed as fold change.

Ursolic acid, cytochalasin B, insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, wortmannin, SB203580, compound C, protease inhibitor and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). High glucose Dulbecco’s modified Eagle’s medium (DMEM) was from Mediatech, Inc. (Cellgro Mediatech, Inc. Manassas, VA). Fetal bovine serum (FBS) was bought from PAA Laboratories (Etobicoke, ON, Canada). Bovine calf serum (BCS) was purchased from Cayman Chemical Company (Ann Arbor, Michigan, USA). The BCA protein assay kit was obtained from Thermo Scientific (San Jose, CA, USA). RIPA lysis buffer was from Millpore (MA, USA). Protein loading buffer was from Bio-Rad (Montreal, QC, Canada). Antibodies against phospho-phosphoinositide-dependent kinase (pPDK), phosphoinositide-dependent kinase (PDK), phospho-protein kinase C (PKC), protein kinase C (PKC), phospho-AS160 (pAS160), AS160, GLUT4, glucose transporter 1 (GLUT1), phospho-phosphoinositide-dependent serine/threonine kinase (pAKT), phosphoinositide-dependent serine/threonine kinase (AKT) and clathrin were from Cell Signaling Technology, Inc. (Beverly, Massachusetts, USA). 2-NBD-glucose was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). BM chemiluminescence blotting substrate kit was from Roche Diagnosis (Laval, QC, Canada).