Fig. 5. GGA clathrin adaptors function specifically during the biosynthetic entry of IRAP into the IRC. (A) Fully differentiated 3T3L1 adipocytes were transfected with 50 µg of IRAP-TfR-WT or IRAP-TfR-AA^76,77 and, 16 hours later, were stimulated with 100 nM insulin. Following surface labeling with the anti-TfR antibody, endocytosis time courses were performed as described in Fig. 1 and in the Materials and Methods. (B) Fully differentiated 3T3L1 adipocytes were electroporated with 50 µg of IRAP-TfR-AA^76,77 and 200 µg of either GGA-WT or VHS-GAT. After an overnight recovery, cells were surface-labeled with the anti-TfR antibody as described in Fig. 3 and in the Materials and Methods. Following a 6-hour washout period at 37°C, cells were treated without or with 100 nM insulin for 30 minutes, fixed, permeabilized and labeled with Texas-Red-conjugated secondary antibody as described under Materials and Methods. The ratio of plasma membrane fluorescence to total fluorescence was determined using the Zeiss LSM software package (mean ± s.e.m. of three independent experiments). a.u., arbitrary units.