FIGURE 7.

IRAP without the lumenal domain is targeted to the donor membranes but has low insulin responsiveness. A and B, undifferentiated (A) and differentiated (B) 3T3-L1 cells stably expressing IRAPtail-EYFP and myc₇-Glut4-ECFP were stained with syntaxin 6 monoclonal antibody and Cy3-conjugated donkey anti-mouse IgG. C, differentiated 3T3-L1 cells stably expressing IRAP-EYFP and IRAPtail-EYFP were treated with 100 nm insulin or carrier (5 μm HCl) for 5 and 15 min at 37 °C and fractionated by differential centrifugation into the plasma membrane (PM) and light microsomal (LM) fractions, which were analyzed by Western blotting. A representative result of five independent experiments is shown. D, differentiated 3T3-L1 cells stably expressing IRAPtail-EYFP and IRAP-EYFP were treated with 100 nm insulin or carrier (5 μm HCl) for 15 min at 37 °C and analyzed by fluorescence-activated cell sorter. Black, IRAP-EYFP (-) insulin; green, IRAP-EYFP (+) insulin; red, IRAPtail-EYFP (-) insulin; blue, IRAPtail-EYFP (+) insulin. A representative result of five independent experiments is shown.