FIG. 6.

ExoT is translocated into the host cell cytoplasm. HeLa cells were infected at an MOI of 5 with PA103ΔUΔT carrying the plasmids shown. After infection for 1.5 h, the medium was removed and separated into supernatant (soluble proteins) and pellet (nonadherent bacteria) fractions. The adherent HeLa cells were lysed and separated into lysate (cytoplasm) and pellet (cell membranes and adherent or internalized bacteria) fractions. Proteins from all fractions were resuspended in SDS-PAGE sample buffer, and 40 μl of each sample (representing 16% of each fraction) was electrophoresed on SDS–8% and SDS–13% polyacrylamide gels. Proteins were electrotransferred to nitrocellulose and hybridized with rabbit polyclonal anti-ExoT antiserum (for 8% gels) or rabbit polyclonal anti-sigma E antiserum (for 13% gels). Goat anti-rabbit IgG–horseradish peroxidase conjugate was used as a secondary antibody, and detection was performed using the ECL system.