Fig. 3. Subcellular distributions of IRAP, HA-GLUT4 and HA-GLUT4 mutants in rat adipose cells. Isolated cells were transfected with 5 µg/ml plasmid DNA and cultured for 24 hours. After harvesting, the cells were incubated without (basal) or with 67 nM insulin for 30 minutes. Then, the cells were fixed, permeabilized, and stained as described in Materials and Methods. Images were collected in single optical sections. (A) Colocalization of IRAP (red) and HA-GLUT4 (green). Squares indicate regions that are magnified in the next row of images. (B) Localization of HA-GLUT4, F5A, 37 and F5A37. Bars, 10 µm.