Synthesis secretion and translocon formation of SseB variants by intracellular Salmonella after infection of RAW macrophages. Macrophages were infected at a MOI of 25 with S. Typhimurium wild type (WT), the sseB strain, or the sseB strain harboring psseB for expression of WT sseB or plasmids for the expression of various mutant alleles of sseB (psseB Δx). At 6 h after infection, the infected cells were fixed with PBS containing 4% sucrose and 4% PFA and solubilized with 0.1% Triton X-100. SseB was immuno-stained using rabbit polyclonal antibody against recombinant SseB as primary antibody and anti rabbit Alexa488 was used as secondary antibody (green). S. Typhimurium was stained with rabbit anti-Salmonella O-1,4,5,12,27 antiserum conjugated with Dylight 547 NHS ester (red). To control the intracellular localization of the bacteria, the late endosomal/lysosomal membrane marker LAMP-1 was immuno-stained using monoclonal antibody and Cy5-conjugated secondary antibody (blue). A) Presence of SseB within the bacterial cytoplasm was investigated by immunofluorescence labeling of SseB after lysozyme permeabilization of intracellular bacteria. B) For analyses of SseB secretion and translocon formation lysozyme treatment was omitted. Note the labeling of SseB in the bacterial cytoplasm for all strain except for the sseB strain in A) and the absence or rare occurrence of punctuated surface labeling for all strains except WT and sseB [psseB] in B).