Organs were collected and homogenized in PBS at 4°C. An aliquot of each homogenate was used to determine its CFU/ml by serial dilution with PBS and plating onto LB agar plates. Each sample was analyzed in triplicate and the analysis
buy PRT062607 was repeated at least three times. The CFU of the sample was expressed as the average of the values obtained. The concentrations of bacteria were recorded as CFU/ml of organ homogenate. The limit of bacteria detection in the organ homogenates was 10 CFU/ml. To prepare protein extracts for Western blot analyses, the homogenates of the spleen click here samples were centrifuged and the pellets that contained the bacteria were resuspended in PBS, following the procedures described previously [16]. All the experimental procedures with animals were MG-132 manufacturer in compliance with the guidelines and policies of the Animal Care and Use Committee (ACUC) of the University of California at Berkeley, and have been approved by the ACUC. Western blot analyses The denatured polypeptides from bacterial lysates were separated on SDS-containing 10-12% polyacrylamide gels cross-linked with N, N”-methylenebisacrylamide (0.05%), transferred electrically to nitrocellulose membranes (Bio-Rad, Hercules, CA), and reacted in an enzyme-linked immunoassay with
a monoclonal anti-FLAG antibody (Sigma, St Louis, MO) and antibodies against Salmonella FliC (BioLegend, San Diego, CA) and DnaK (StressGen, Victoria, British Columbia, Canada), followed by an anti-mouse IgG conjugated with alkaline phosphatase [16, 36]. The membranes were subsequently stained with a chemiluminescent substrate with the aid of a Western chemiluminescent substrate kit (Amersham Bcl-w Inc, GE Healthcare) and quantified with a STORM840 phosphorimager. Normalization of samples was also carried out by loading total proteins extracted from the same CFU (e.g. 5 × 107 CFU) of bacteria in each lane. Acknowledgements We thank Cindy Loui, Yong Bai, Hongwei Gu, and Huiyuan Jiang for suggestions and excellent
technical assistance. K. K., G. V., and E. Y. were partially supported by a Block Grant Predoctoral Fellowship (UC-Berkeley). The research has been supported by grants from USDA (CALR-2005-01892) and NIH (RO1-AI041927 and RO1-AI014842). References 1. Ohl ME, Miller SI: Salmonella : a model for bacterial pathogenesis. Annu Rev Med 2001, 52:259–274.PubMedCrossRef 2. Pang T, Levine MM, Ivanoff B, Wain J, Finlay BB: Typhoid fever–important issues still remain. Trends Microbiol 1998,6(4):131–133.PubMedCrossRef 3. Altekruse SF, Swerdlow DL: The changing epidemiology of foodborne diseases. Am J Med Sci 1996,311(1):23–29.PubMedCrossRef 4. Galan JE: Salmonella interactions with host cells: type III secretion at work. Annu Rev Cell Dev Biol 2001, 17:53–86.PubMedCrossRef 5. Galan JE, Wolf-Watz H: Protein delivery into eukaryotic cells by type III secretion machines. Nature 2006,444(7119):567–573.PubMedCrossRef 6. Imlay JA: Pathways of oxidative damage.
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