2.7. GLUT4 Translocation Assay

The surface GLUT4 level in non-permeabilized L6-GLUT4myc myotubes was measured by an antibody-coupled colorimetric assay [29]. After indicated treatments with BSD, myotubes were fixed in 3% paraformaldehyde and quenched in 100 mM glycine. After the blocking in 5% FBS, cells were incubated with the anti-myc primary antibody solution for 1 h followed by washing with PBS to remove excess labeling. The bound antibody was probed by HRP-conjugated secondary antibodies followed by the detection of bound HRP by an O-phenylenediaminedihydrochloride reagent.