2.9. Western blot
Cell lysates containing 30 μg of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then, it was transferred to nitrocellulose sheet in a western blot apparatus (Bio-Rad, Hercules, CA,
USA). The nitrocellulose sheet was blocked with 5% skim milk for 1 h, rinsed by Tris-buffered-saline with Tween (1mol/L Tris, 5 mol/L NaCl, and 0.1% Tween 20), and incubated for overnight with primary antibodies of iNOS and GLUT4.
Nitrocellulose membrane was again washed with Trisbuffered-saline with Tween followed by incubation with
secondary antibody (horseradish peroxidase-conjugated IgG 1:2000) for 1 h. Finally, the expressed proteins were measured
by analyzing the signal captured in nitrocellulose membrane
using chemiluminescent substrate and VisionWorksTMLS
(Analysis software, Upland, CA, USA).