Flagellin co-localizes with endosomal and lysosomal markers.

<p><b>A</b>. Following AP exposure to Caco-2BBe cells, GFP-labeled flagellin (green) was internalized and, within 15 min, co-localized with an antibody that recognized an early endosomal surface marker (EEA1) and secondary antibody (red) as shown in the middle z-sections by confocal microscopy. Lower right panel shows merged images and indicates co-localization of flagellin with EEA1.Cell nuclei were stained with DAPI (blue). <b>B</b>. Within 1 h flagellin (green) co-localized with the lysosome marker LAMP-1 (red). The confocal images shown are representative of 3 independent experiments. <b>C</b>–<b>F</b>. 6HIS-Flagellin was added to the AP or BL side of polarized Caco-2BBE cells at 19°C allowing traffic up to (but not beyond) the recycling endosome. In some experiments biotinylated transferrin also was added to the apical surface as a marker for the endosomal pathway. Cells then were either fractionated (see methods) without allowing further traffic or warmed to 37°C for the times indicated prior to fractionation. NOTE: White arrows indicate flagellin protein in A and B. <b>C</b>. Intracellular localization of internalized flagellin protein (normalized for total cell-associated protein) following incubation at 19°C. <b>D</b>. Western blots of endosomal fractionation as described in C and in the methods. <b>E</b>. Intracellular localization of 6HIS-Flagellin when internalization of protein at 19°C was followed by incubation at 37°C allowing traffic beyond the recycling endosome. <b>F-G</b>. Western blots of fractionation as described in E and in the methods.