siRNA-mediated EHD protein knock-down effects on transferrin recycling. Following EHD protein knock-down, transferrin loading was carried out essentially as in Figure 10 except that Transferrin-coupled Alexa Fluor 488 was loaded for 30 min. Following transferrin loading, cells were washed with ice-cold PBS, changed to serum-containing media and allowed to recycle transferrin at 37°C for various time points. The cells were fixed and scanned using a confocal microscope equipped with a 40× objective lens. Knock-down cells retaining more transferrin than irrelevant controls at 60 min of chase were considered to be delayed in recycling transferrin. Irrelevant siRNA mildly affected transferrin recycling at 30 min of chase as compared to mock. Bar, 10 μm.