Differential effects of wild type and ΔEH mutants of EHD1, 3, & 4 versus EHD2 on transferrin exit from the ERC. Untransfected HeLa cells or cells transiently transfected with Myc-EHD1 or Myc-EHD1 ΔEH were loaded with Transferrin-coupled Alexa Fluor 488 (Tf, green) in internalization buffer at 37°C for 30 minutes 24 h after transfection, washed with ice-cold PBS and chased with serum-containing medium. The cells were fixed at various time points, stained with antibodies for Myc (9E10, red) and scanned on a confocal microscope. Similar experiments with EHD2-4 and EHD2-4 ΔEH are shown in Additional File 5. (B) Cells expressing Myc-EHD proteins (black bars) or Myc-EHD ΔEH proteins (grey bars) from a representative experiment were counted with respect to Tf retention after 60 min of chase. At least 200 cells were counted in each case.