HeLa cells were transfected with pcDNA-prME, pcDNA-prME-opt or a pcDNA empty vector using calcium phosphate precipitate method. Two days after transfection, cells were detached by incubation in 10 mM EDTA at 37uC for 10 min, fixed in 2% paraformaldehyde, and then permeabilized in 0.1% Triton X-100. After washing, the cells were incubated with a mouse anti-E antibody (4E11 