Peritoneal macrophages were allowed to adhere to sterile glass coverslips overnight. At the indicated time points after infection, cells were fixed in 3% paraformaldehyde in PBS for 20 min at RT. Staining of L. monocytogenes was performed as previously described (50); extracellular L. monocytogenes were excluded from the analysis. Nuclei were stained with 4′,6-diamidino-2-phenylindole and NF-κB p65 with a monoclonal antibody (catalog no. sc-8008, Santa Cruz Biotechnology). Samples were mounted on glass microscopic slides in ProLong Gold antifade reagent (Invitrogen) and analyzed using an IX81 fluorescence microscope (Olympus). In each experiment, at least 100 infected cells per time point and condition were analyzed.

For visualization of FLAG-tagged proteins, macrophages were transfected and fixed as described above. Macrophages were permeabilized with 0.25% Triton X-100. After blocking with 10% bovine serum albumin in PBS, FLAG-tagged proteins were stained using a monoclonal antibody recognizing FLAG (1:1000; catalog no. F1804, Sigma-Aldrich) at 37°C for 2 hours. Samples were mounted on glass microscopic slides and analyzed as described above.

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