FIG. 7.

eGFP-Eps15ΔEH2,3 and eGFP-GPRL-amphiphysin1 block transferrin uptake in THP-1 cells. THP-1 cells (15 × 10⁶) were transfected by nucleofection with eGFP-tagged plasmids encoding Eps15ΔEH2,3, Eps15ΔAP2, amphiphysin 1 (Amph1), and Amph1-GPRL for 24 h. The eGFP-sorted cells were starved of serum in Dulbecco modified Eagle medium-BSA for 4 h, incubated in the cold with mouse anti-transferrin receptor monoclonal antibodies and anti-mouse IgG coupled to 5-nm-diameter colloidal gold, warmed for 30 min, and processed for TEM (for the complete experiment, also see Fig. 6 and its legend). The numbers of gold particles on the plasma membrane (arrows) and on endosomal membranes (arrowhead in panel c) were determined, and the amounts of endosomal gold represented in panel d are percentages of the total, including the number of gold particles (n) and the number of cells analyzed.