Fig. 3. Intracellular transport of Salmonella in MBSA-MDP-treated cells. (A) Salmonella-containing early phagosomes were purified from untreated cells as well as from MDP and MBSA-MDP-treated cells as described in the Materials and Methods. Proteins from the respective phagosomes (40 µg each) were separated by SDS-PAGE and transferred to nitrocellulose membranes. After incubation with appropriate dilutions of specific antibodies, the proteins on the membranes were visualized using an appropriate HRP-labelled second antibody using ECL. Results from western blots are representative of three independent preparations. (B) In vitro fusion of early endosomes with Salmonella-containing phagosomes isolated from untreated or MBSA-MDP-treated cells. Fusion between early endosomes with respective Salmonella-containing phagosomes isolated from untreated or MBSA-MDP treated cells was carried out either in the presence of normal cytosol or cytosol prepared from MBSA-MDP-treated cells as indicated. Fusion was measured as described in the Materials and Methods. Maximum fusion between endosomes and phagosomes isolated from untreated control cells was observed at 0.5 mg/ml of normal cytosol concentration, which was normalized to one unit. The results are expressed as relative fusion from three independent experiments±s.d. One unit corresponds to 10.2 ng of HRP activity/mg of protein. (C) Intracellular transport of live Salmonella to the lysosomes in MBSA-MDP-treated cells. To determine the transport of Salmonella to the lysosomes by MBSA-MDP-treated cells, J774E cells (1x10⁶ cells) were treated with MDP in the free or conjugated form for 12 hours at 37°C as described earlier. Subsequently, J774E macrophages were preloaded with avidin-HRP and chased for 90 minutes to label the lysosomes. Cells were pulsed with live or dead biotinylated Salmonella at 37°C for a short period of time (5 minutes) to restrict their entry to the early compartment followed by a chase as described in the Materials and Methods. At indicated times the formation of the bacteria biotin-avidin-HRP complex was measured to determine the transport of the Salmonella to lysosomes. Each point represents the mean±s.d. from three independent experiments and is expressed as relative units of transport to lysosomes.