GLUT4 and CD36 on the cell surface were determined via a modified biotinylation method described previously (3). Briefly, isolated soleus muscle or L6 myotubes were stimulated with or without insulin for 50 min and then incubated in KRB buffer (±insulin) containing Sulfo-NHS-SS-Biotin (1 mg/ml) (Thermo Fisher Scientific) for 30 min. After biotinylation of cell surface proteins, the soleus muscle or L6 myotubes were rinsed with 50 mM tris-HCl buffer (pH 8.0) twice to remove free Sulfo-NHS-SS-Biotin. After lysis, tissue/cell lysates were incubated with NeutrAvidin Agarose (Thermo Fisher Scientific) at 4°C overnight. After incubation, the beads were washed three times with the ice-cold phosphate-buffered saline buffer containing 1% NP-40 to remove nonspecific binding proteins. Afterward, biotinylated cell surface proteins were eluted in SDS sample buffer at room temperature for 30 min and subjected to immunoblotting analysis. Biotinylated GLUT4 and CD36 were detected using corresponding specific antibodies.

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