RF/6A cells were transfected with GFP–N-WASP, and at 23 h posttransfection, fresh phenol red-free Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum was added, and cells were incubated with flash red beads (0.51-µm diameter; Bangs Laboratories) coated with rEtpE-C (11 (link)). Samples were moved to a live-cell imaging chamber at 37°C connected to a TI-E inverted research microscope (Nikon Instruments) controlled by Nikon Elements software and equipped with a spinning-disk confocal unit (Yokogawa Electric), a 100× objective lens (Plan Apochromat Lambda, NA 1.45; Nikon), and an electron multiplying charge-coupled device (EMCCD) camera (iXon DU897 Ultra; Andor Technology). Three-dimensional confocal image stacks were acquired at 1-min intervals for 2 h, with a step size of 0.1 µm along the z axis. The bead internalization movie (see Movie S1 in the supplemental material) was prepared with ImageJ software by creating a stack of confocal images acquired at the upper surface of an RF/6A cell.