Fig. 5. Reduced Tf recycling in cells overexpressing SARA. (A) HEK cells transiently transfected with GFP-SARA were loaded with Cy3-Tf at 37°C for 2 hours and chased in the presence of excess unlabeled Tf and desferroxamine for 0, 10, and 30 minutes before the cells were chilled, fixed and observed. At all time points, Cy3-Tf was mainly accumulated in the GFP-SARA positive endosomal compartments (arrows). (B) HEK cells double-transfected with GFP-SARA and Rab5S34N were preloaded with Cy3-Tf for 2 hours and chased for 30 minutes. The double transfected cells (indicated by cytosolic GFP signal) release Cy3-Tf in a manner indistinguishable from the neighboring cells. Bar, 10 µm. (C) The ¹²⁵I-labeled Tf was loaded on HEK stably expressed SARA or HEK cells for 2 hours before the chase. The natural logarithm of the percent of Tf remaining inside the cell versus the chase time from one experiment is shown. (D) The mean±s.e.m. of the recycling rate constants from three independent experiments are presented. The Tf recycling rate constant of the SARA stable line is significantly slower than that of HEK control cells (P<0.01, two-tailed Student's t test).