2.9. GLUT4 translocation assay

GLUT4 exocytosis was measured as previously described [26]. Briefly, L6-G4-myc and L6 myoblasts were cultured and transfected as described above. Following three hours of serum starvation, cells were stimulated with insulin (100 nM) for 10 min before being washed with ice-cold PBS, fixed with 3% paraformaldehyde for 10 min, blocked with 3% goat serum and incubated with polyclonal anti-Myc-Tag antibody (1:200) for 60 min at 4 °C. Following primary antibody incubation, cells were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:2,000) for 60 min at 4 °C. Cells were then washed with PBS and incubated with o-phenylenediamine dihydrochloride (OPD) for 20–30 min at room temperature. Incubation was stopped with 3 M of HCL and absorbance of the supernatant measured at 492 nM using a ThermoFisher Multiskan spectrophotometer (Thermofisher, Waltham, MA, USA). Background myc-tag binding was determined from L6 myoblasts that do not express the myc-tagged GLUT4 and subtracted from appropriate values.