The protocols used were in compliance with the guidelines and

policies of the Animal Care and Use BI 10773 in vitro Committee (ACUC) of the University of California at Berkeley. Overnight bacterial cultures were serially diluted to suitable CFU/ml in PBS for infection. To assess the virulence of the tested strains, groups of five mice were either inoculated intragastrically with 5 × 106CFU per BALB/c mouse and 1 × 103CFU per SCID mouse or intraperitoneally selleck with 1 × 102CFU per BALB/c mouse and 1 × 101CFU per SCID mouse. Mice were monitored during the course of infection, and those animals that exhibited extreme stress or became moribund were euthanized [45,48]. For organ colonization andin vivoexperiments, groups of five mice were inoculated intraperitoneally with 1 × 105or 1 × 107CFU per BALB/c mouse or 1 × 102or 1 × 104CFU per SCID mouse of the bacterial strains, and were euthanized at 5 days or 18 hours after inoculation, respectively. Mice (5 animals per group) were also inoculated intragastrically with 1 × 105or 1 × 108CFU per BALB/c mouse or 1 × 102or 1 × 104CFU per SCID mouse of the bacterial strains and were euthanized at 7 days or 24 hours after inoculation, respectively. Organs

were collected and homogenized in cold PBS. An aliquot of homogenate was used to determine its CFU/ml by serial dilution with PBS and plating on LB agar plates [45,48]. To prepare protein extracts for Western GSK2126458 molecular weight analyses, the homogenates of the spleen samples were centrifuged at 9,000 × g and 4°C for 10 minutes. The pellets from the spleen were resuspended in 0.5 ml of cold lysis buffer (120 mM NaCl, 4 mM MgCl2, 20 mM Tris/HCl, pH 7.5, 1% Triton-X100) supplemented with protease inhibitors (complete EDTA-free cocktail, Roche), incubated at 4°C for 1 hour, centrifuged at 18,000 × g and 4°C for 10 minutes. The pellets that contained the bacteria were

resuspended in PBS for Western analyses Phosphoprotein phosphatase [45,48]. For the cecum samples, the homogenates were incubated on ice for 10 minutes. The upper clear suspensions were transferred and centrifuged at 15,000 × g and 4°C for 10 minutes. The pellets were washed in PBS, centrifuged at 18,000 × g and 4°C for 10 minutes, and resuspended in PBS for Western analyses [45,48]. Western analyses The denatured polypeptides from bacterial lysates were separated on SDS-containing 10–12% polyacrylamide gels cross-linked withN,N”"-methylenebisacrylamide, transferred electrically to nitrocellulose membranes, and reacted in an enzyme-linked immunoassay with anti-mouse IgG conjugated with alkaline phosphatase in addition to the antibodies against the FLAG sequence (Sigma, St Louis, MO) andSalmonellaDnaK protein [45,49]. The membranes were subsequently stained with a chemiluminescent substrate with the aid of a Western chemiluminescent substrate kit (Amersham Inc, GE Healthcare) and quantitated with a STORM840 phosphorimager. Quantitation was performed in the linear range of protein detection.