, 2005). Finally, effector proteins were immunodetected as described below. Human laryngeal epithelial (HEp-2) cells (ATCC, CCL-23) were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Infected monolayers (multiplicity of infection=10 : 1) were incubated for 20 min at 37 °C in 5% CO2, washed twice with PBS and then incubated in fresh tissue-culture medium containing 100 mg mL−1 of gentamicin for 30 min to remove extracellular bacteria. At 20 min and 24 h postinfection monolayers were washed twice with cold Hank’s balanced salt solution (HBSS) and lysed with 1.0 mL of HBSS containing 0.1% Triton X-100 and 1 mM

phenylmethylsulfonyl fluoride as described by Kubori & Galán (2003). This Nivolumab procedure lyses the infected cells but does not affect the integrity of the bacterial membrane (Collazo & Galán, 1997). An aliquot of this suspension was used to determine the number of intracellular bacteria by plating serial dilutions onto LB agar plates. Cell lysates were collected in chilled microfuge tubes, and centrifuged at 17 000 g for 15 min at 4 °C to separate the soluble fraction, containing bacterial proteins that have been translocated into the host cell cytosol, from the insoluble fraction, which contains the internalized bacteria. The LY294002 soluble fraction was filtered through a 0.45-μm-pore size filter and subjected to 10% trichloroacetic

acid precipitation and sedimented by high-speed centrifugation (14 000 g for 30 min). The pellet was washed in cold acetone and resuspended in PBS and Laemmli buffer. The insoluble fraction was washed once with cold PBS and resuspended in an appropriate volume of PBS and Laemmli buffer. The protein extracts were boiled for 5–10 min, and resolved on SDS-PAGE. Finally, effector

proteins were immunodetected using mouse-monoclonal anti-FLAG M2-peroxidase (HRP) antibodies (Sigma, St Louis, MO). Some blots were reprobed with rabbit-polyclonal antibodies to actin (Sigma) as cytosolic protein marker. Detection was performed by chemiluminiscence (Luminol, Santa Cruz Biotechnology, Santa Cruz, CA). Six to 8-week-old BALB/c mice were purchased from the Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina and kept in Evodiamine our animal house throughout the experiments. All experiments were performed in accordance with the guidelines of the University of Buenos Aires School of Medicine Animal Care and Use Committee. Groups of six mice were inoculated intraperitoneally with 100 μL of serial dilutions of the bacterial suspension. Survival of infected mice was recorded for a minimum of 4 weeks. LD50 was calculated as described by Reed & Muench (1938). In vivo expression of SopB was studied daily after infection. Mice were inoculated intraperitoneally with four different lethal doses of Salmonella-tagged strains (107, 106, 104 and 102 CFU per mouse); thus a comparable number of bacteria was recovered from MLN at all time points.