After ultracentrifugation, RSPs were resuspended in 100 ml PBS to which 33 ml of 4X NuPAGE LDS (lithium dodecyl sulfate) sample buffer (Invitrogen) was added. RSPs were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4,12% NuPAGE Novex Bis-Tris Gels (Invitrogen). For analysis of cell lysates by SDS-PAGE, HeLa-prME cells were detached by incubation in 10 mM EDTA at 37uC for 10 min and resuspended in 500 ml of PBS. Cell suspensions were then mixed with 167 ml 4X of sample buffer and then sonicated before electrophoresis of samples as described above. For immunodetection, proteins were blotted from gels onto polyvinylidene difluoride (PVDF) membranes. The membrane was blocked overnight in 5% milk in PBST solution and then incubated with anti-E antibody (4E11, 1:1000) for 1 hour. After washes, the membrane was incubated for 1 hour at room temperature with a horseradish peroxidase-labeled goat anti mouse IgG polyclonal antibody. The membrane was finally visualized using ECL Western blot detection reagents (Invitrogen) and Amersham Hyperfilm ECL (GE Healthcare, Waukesha, WI, USA). For silver staining, the gel was fixed for 30 minutes and incubated with sodium thiosulfate for 30 minutes at room temperature. After three washes, the gel was incubated with Silver Nitrate for 40 minutes and developed for 15 minutes in sodium carbonate solution (25 g/L). EDTA solution (40 mM) was used to stop the development.