As SseB moves away from the <i>Salmonella</i> surface, it is associated with the vacuolar membrane.

<p><b>(A)</b> RAW264.7 macrophages were infected with WT <i>Salmonella</i> harboring pFPV25.1 for the constitutive expression of GFP (green). Cells were fixed, permeabilized, and immunostained for mouse monoclonal anti-LAMP-1 (blue) and rabbit polyclonal anti-SseB (red) followed by Alexa568- or Alexa647-labeled secondary antibodies, respectively, for various times as indicated. The merged image indicates that once SseB was detached from the bacterial cell surface, it was co-localized with the host endosomal membrane (i.e., LAMP-1–positive membranes). Images were obtained using the Nikon A1R confocal microscope. Localization resulted in magenta staining of <i>Salmonella</i> SseB as analyzed by Image J software. Scale bar = 3μm. <b>(B)</b> Results of three independent experiments in which macrophages were infected with WT GFP-expressing <i>Salmonella</i> and immunostained with LAMP-1 and SseB at 3.5 h, 4 h, 5 h, and 6 h post-infection. The values represent the mean ± SEM.