A- IR, IRS-1 and GLUT-4 expression in total extract from skeletal muscle; B- IR protein content and IR tyrosine phosphorylation (pIR); C- Membrane-associated GLUT-4 content. Cellular proteins (30 μg total) were subjected to 10% SDS-PAGE, transferred to PVDF filters and blocked with Tween-TBS containing 1% BSA. Primary antibodies used in Western analysis were anti-insulin receptor β-subunit (IR); anti-IRS-1; anti-GLUT-4 and anti-phosphotyrosine. PVDF filters were next incubated with appropriate secondary antibody conjugated to biotin followed by 1-h incubation with horseradish peroxidase-conjugated streptavidin. Immunoreactive proteins were visualized by DAB staining.