An MOI of ,5:1 was used to infect BMDMs (1610 7 L. monocytogenes and 2610 6 BMDMs). Cell samples were rinsed once with PBS containing calcium and magnesium and lysed with 2X Laemmli sample buffer (125 mM Tris HCl pH 6.8, 4% sodium dodecyl sulfate, 30% glycerol, 1% b-mercaptoethanol, 0.005% bromophenol blue). The Laemmli buffer was added directly to the infected cells and scraped off the tissue culture treated Petri dishes using a cell scraper. The samples were collected into Eppendorf tubes, boiled for 5 minutes at 95uC and loaded on 12% pre-cast NuPage gels (Invitrogen, Carlsbad, CA). Gels were run in 1 X 3morpholinopropane-1-sulfonic acid (MOPS) buffer (Invitrogen). Gels were transferred to polyvinylidene fluoride (PVDF) membrane at 15V for 1 hour on a Trans-Blot Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA). The membrane was blocked for 1 hour at room temperature with Odyssey blocking buffer (LI-COR, Nebraska, NE, catalogue no. 929-97301) diluted 1:1 with PBS. The membrane was incubated overnight at 4uC with mouse monoclonal anti-LC3 antibody (courtesy of R. Kopito and B. Riley, Stanford University) at 1:500 in blocking buffer containing 0.02% Sodium Azide and 0.1% Tween-20. The membrane was washed three times for 10 minutes with TBST. The membrane was incubated with the LI-COR secondary antibody covalently attached to an infrared-emitting fluorophore IRdye-680 and IRdye-800 (LI-COR Biosciences, Lincoln, NE (goat anti-mouse IgG, 0.5 mg)) for 1 hour at room temperature. The membrane was washed three times for 10 minutes with TBST followed by one wash with 1X PBS. Membranes were scanned at 700 nm or 800 nm using the Odyssey Infrared Imaging System (LI-COR Biosciences), and quantitated using the accompanying software package (Odyssey V 3.0). Anti-gamma-tubulin, mouse monoclonal (clone GTU-88, Sigma catalogue no. T6557) was used as a loading control. Westerns were repeated three independent times.