Fig. 1. Newly synthesized IRAP-TfR-AA^76,77 defaults to the plasma membrane, where it accumulates under steady-state conditions. (A) Differentiated 3T3L1 adipocytes were electroporated with 50 µg of IRAP-TfR-WT (panels a and c) or IRAP-TfR-AA^76,77 (panels b and d) and, 16 hours later, cells were serum-starved and treated without (panels a and b) or with (panels c and d) 100 nM insulin for 20 minutes. The cells were then fixed, permeabilized and labeled with an anti-TfR antibody followed by Texas-Red-conjugated secondary antibody, and were then visualized with a Zeiss LSM510 scanning laser confocal microscope. (B) The data in A were quantified as the ratio of surface fluorescence:total fluorescence, as described in the Materials and Methods. White bars, basal cells; black bars, insulin-stimulated cells. (C) Differentiated 3T3L1 adipocytes were electroporated with 50 µg of IRAP-TfR-AA^76,77 and, 16 hours later, cells were incubated with vehicle alone (panels a-d) or vehicle plus 10 µg/ml cycloheximide (CHX) (panels e-h) for 0, 1, 2 or 4 hours. The cells were then processed for confocal microscopy as indicated in A. (D) The data presented in C were quantified by calculating the ratio of plasma membrane:total fluorescence, as described under Materials and Methods. Results are from three independent experiments, with fifteen cells per experiment (mean ± s.e.m.). (E) Fully differentiated 3T3L1 adipocytes were transfected with 50 µg IRAP-TfR-WT or 50 µg IRAP-TfR-AA^76,77 cDNA. After an overnight recovery period, cells were incubated for 0, 0.5, 1, 2, 4 or 6 hours in 10 µg/ml CHX. In lane 1, cells were plated immediately in CHX after electroporation and were then incubated for 6 hours. At the indicated time points, cell lysates were prepared and subjected to western blotting. An anti-IRAP antibody was used to detect both endogenous IRAP (upper panel) and the expressed IRAP-TfR chimera (lower panel). a.u., arbitrary units.