Fig. 6. Overexpression of GFP-Rab11wt and mutants inhibits transferrin recycling. Cells were first pre-incubated for 1 hour at 37°C in serum-free RPMI medium supplemented with 1% BSA to deplete endogenous transferrin (Tf) and then were allowed to internalize ¹²⁵I-labeled human Tf continuously for 1 hour at 37°C. Cells were washed at 4°C, and surface-bound transferrin was dissociated by acid treatment as described in the Materials and Methods. Cells were then further incubated at 37°C in serum-free RPMI medium containing 100 µM deferoxamine mesylate and 20 µg/ml unlabeled human Tf. At different times, the incubations were stopped by adding ice-cold PBS and placing the samples on ice. The samples were centrifuged to sediment the cells, and the medium was collected. The cell pellet was washed with the acetate buffer to remove surface-bound Tf. The radioactivity in both the medium and the cell pellet was determined by -counting. Data represent the mean±s.e.m. of three independent experiments.