Membrane topology of translocated SseF.

<p>HeLa cells were infected with the <i>sseF</i> strain harboring plasmids for the expression of HA-tagged alleles of <i>sseF</i>. At 16 h after infection, the cells were fixed and subjected to permeabilization by saponin or digitonin as indicated. Saponin treatment allows the penetration of antibody across the cytoplasmic and endosomal membranes of the host cell, while digitonin treatment only permeabilizes the cytoplasmic membrane. To control the selectivity of the permeabilization, intracellular <i>Salmonella</i> were immune-stained with antibody against LPS (white). Detection of translocated SseF was performed with antibody against the HA tag (green). As control for a cytosolic protein, host cell β-tubulin was labeled (red). In order to locate intracellular bacteria, DAPI staining of bacterial DNA was performed and the location of DAPI-stained bacteria is indicated by arrows for the digitonin experiment (DAPI staining not shown).