P ampk

Thoracic aorta and muscle homogenates were prepared in ice-cold buffer containing 250 mM sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 20 mM potassium phosphate buffer (pH 7.6). The homogenates were centrifuged at 8,000 rpm for 10 min at 4°C, after which the resulting supernatants were centrifuged at 13,000 rpm for 5 min at 4°C to produce a cytosolic fraction for protein analysis. The recovered proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, which were blocked with 5% bovine serum albumin (BSA) powder in 0.05% Tween 20-Tris-buffered saline (TBS-T) for 1 h. Specific primary antibodies against ICAM-1, VCAM-1, E-selectin, eNOS, ET-1 (in aorta), AMP-activated protein kinase (AMPK), p-AMPK, and insulin receptor substrate-1 (IRS-1) (in muscle) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The nitrocellulose membranes were incubated overnight at 4°C with protein antibodies. The blots were washed several times with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h after which immunoreactive bands were visualized using an enhanced chemiluminescence system (Amersham, Buchinghamshire, UK). The bands were analyzed densitometrically using a Chemi-doc Image Analyzer (Bio-Rad, Hercules, CA, USA).