siRNA-mediated EHD protein knock-down effects on transferrin loading in HeLa cells. (A) HeLa cells were seeded on autoclaved glass coverslips in 6-well plates for 24 h followed by transfection of 200 pmol of double-stranded RNA oligonucleotides with irrelevant or EHD siRNA for 48 h prior to transferrin loading. Cells were starved for 30 min in starvation media followed by Transferrin-coupled Alexa Fluor 594 in internalization buffer at 37°C for 15 min, washed with ice-cold PBS, fixed and scanned using a confocal microscope equipped with a 40× objective lens. The arrows in the EHD1 siRNA depict an ERC transferrin loading phenotype while the arrows in the EHD4 siRNA depict an EE phenotype. (B) Cells were transfected with siRNA for EHD proteins for 24 h and further transfected with Rab5-GFP or Rab11-GFP for an additional 24 h. Cells were then loaded with labeled transferrin for 15 min as described in Methods. Arrowheads point to colocalized structures. Bar, 10 μm. Data are representative of 3 individual experiments.