FIGURE 6.

HRG-1 overexpression regulates receptor trafficking. A, MCF-7 cells stably overexpressing HA-HRG-1 were assessed for cellular levels of TfR by Western blotting. The data represent the average of TfR levels from three experiments (n = 3; *, p < 0.05 using t test). B, TfR internalization was compared in MCF-7/HA and MCF-7/HA-HRG-1 cells. Cells were serum-starved for 1 h, followed by incubation at 4 °C with 25 μg/μl Tf-Alexa488 for 90 min in triplicate wells. Prewarmed media were added to the cells, which were transferred to 37 °C to induce TfR/Tf-Alexa488 internalization. At the indicated time points, cells were transferred to ice. Noninternalized ligand/receptors were removed by an acid wash, and internalized TfR/Tf-Alexa488 was quantified by FACS. The data are represented as the percentage of internalized receptor compared with cells maintained at 4 °C whereby receptor internalization is inhibited. *, p < 0.05 using t test. Panel shows relative cell surface TfR levels in MCF-7/HA and MCF-7/HA-HRG-1 cells, determined by FACS quantification of surface-bound Tf-Alexa488 as described for Fig. 3E.