FIG. 4.

Iodixanol equilibrium gradient sedimentation analysis of internalized Tf. 3T3-L1 adipocytes were serum starved in DMEM for 16 h and then incubated at 37°C for 2 min with KRP buffer containing ¹²⁵I-diferric human Tf (∼1.0 μCi/ml) and BSA (1 mg/ml). As a control to estimate nonspecific uptake, duplicate plates were incubated in the presence of excess (20 μM) unlabelled holo Tf. After two quick washes, the cells were incubated at 37°C for 0 (●), 3 (○), and 20 (▴) min to allow Tf internalization. Cells were washed and subjected to subcellular fractionation and iodixanol sedimentation analysis as described in Materials and Methods. Fractions were counted in a gamma counter, and internalized Tf was quantitated by subtracting background values from each fraction. The results shown are representative of two separate experiments.