The frozen skeletal muscle was weighed, defrosted on ice and lysed in lysis buffer (800 μL) (Pierce Biotechnologies, Rockford, CA, USA) using a tissue lyser at 25 Hz. Thereafter, the samples were centrifuged for 20 min at 12,000 rpm at 4 °C in a micro centrifuge. The supernatant was collected and kept in a fresh tube at − 20 °C until required. Protein (30 µg) was mixed with an equal volume of 2× Laemmli sample buffer (containing β-mercaptoethanol), before it was denatured at 95 °C. The denatured protein sample (30 µg) was loaded onto a 12% Mini-protein TGX Precast Gels, 10 well comb, 50 µL/well (BioRad), along with 12 µL BioRad-Western C molecular weight marker. The gel was run for 1 h at 150 V in Tris/Glycine/SDS-PAGE Buffer 1× and then transferred to a polyvinylidene fluoride membrane (PVDF, Bio-Rad, Hercules, CA, USA) [
19]. Membranes were incubated at 4 °C for 16 h with different primary antibodies which include IRS-1
Ser307, p-AKT and p-GSK-β, [Cell Signaling Technology, Danvers, MA, USA; (1:500), (1:1000), (1:1000) respectively], GLUT 4 [Sigma-Aldrich Chemical Co., St. Louis, MO, USA, (1:1000)]. β-Actin [Cell Signaling Technology, Danvers, MA, USA, (1:4000)] antibody was added as a loading control. The membrane was incubated with an appropriate horseradish peroxidase conjugated secondary antibody (1:4000) in blocking buffer (2.5% milk) at room temperature for 90 min, and then placed on a plastic sheet and 2 ml chemiluminescent substrate (1:1 of luminal solution and reaction buffer) was added to the membrane for signal development. The proteins were detected using BioRad ChemiDoc and the signaling intensity of the bands was quantified using the image J software.