Fig. 2. Impaired recycling in the absence of EHD1 leads to accumulation of 1 integrin in the endosomal recycling compartment. The intracellular localization of labeled transferrin and 1 integrins was compared using pulse-chase analysis in HeLa cells treated for 48 hours with mock-RNAi (A-C) or EHD1-RNAi (D-F). Cells were pulsed with 12G10 antibody that preferentially recognizes ligand-bound human 1 integrin (MCA2028; Serotec) for 2 hours, acid-stripped to remove non-internalized antibodies and chased for 30 minutes at 37°C. During this chase, transferrin–Alexa-Fluor-568 (Tf-568) was added for 15 minutes, and the Tf-568 was then chased in complete media for the remaining 15 minutes prior to fixation. Permeabilized cells were incubated with Alexa-Fluor-488-conjugated donkey anti-mouse antibody and analyzed by z-sectioning; confocal images were obtained every 0.4 µm. The representative images show the same z-sections through mock- and RNAi-treated cells, and crosshairs show colocalizations on x- and y-axes. Arrows highlight specific vesicular membrane structures containing both transferrin and 1 integrins. Bar, 10 µm.