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Fig. 2. IRAP-TfR-AA^76,77 displays a normal rate and extent of plasma membrane endocytosis. Differentiated 3T3L1 adipocytes were transfected with 50 µg of IRAP-TfR-WT or IRAP-TfR-AA^76,77 and, 16 hours later, stimulated with 100 nM insulin (this is only necessary to induce TfR-IRAP-WT translocation but was done for both constructs for internal consistency). The cells were then cooled to 4°C and the cell surface was labeled with the anti-TfR antibody. The cells were washed and warmed to 37°C for various times, as indicated. The cells were then fixed, permeabilized and labeled with Texas-Red-conjugated anti-TfR antibody. (A) Representative cell images are shown. (B) The percentage of cell-surface signal was calculated using the Zeiss LSM 510 software package (mean ± s.e.m. of three independent experiments). (C) Fully differentiated 3T3L1 adipocytes were co-electroporated with 50 µg of both HA-GLUT4 and IRAP-TfR-AA^76,77, and were allowed to recover overnight. Cells were then stimulated with 100 nM insulin for 20 minutes, and then surface-labeled on ice with a mouse monoclonal anti-TfR antibody and a polyclonal rabbit HA antibody for 60 minutes. After extensive washing with ice-cold PBS, cells were either fixed in paraformaldehyde (t=0) or were warmed to 37°C for 6 hours (t=6 h), fixed and labeled with Texas-Red-conjugated donkey anti-mouse and Alexa-Fluor-488-conjugated donkey anti-rabbit secondary antibodies. Images were collected on a Zeiss LSM 510 META confocal microscope. a.u., arbitrary units.

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