For preparation of cell lysates, cells were washed once with cold

For preparation of cell lysates, cells were washed once with cold PBS buffer, resuspended in TES buffer to 10% of the original

volume of culture. For Hbl B overexpressing strains, cells were lysed by mechanical disruption using Lysing Matrix B (MP Biomedicals) in a Mini-BeadBeater-8 click here (BioSpec) according to manufacturer’s specifications. For mutant strains and azide-treated cultures, cells were lysed by incubation at 37°C for 60 minutes with 1 mg ml-1 lysozyme, followed by six rounds of freezing and thawing. All samples were used within 2 weeks and all experiments were performed at least twice. Analysis of samples Protein electrophoresis was performed using the NuPAGE Novex Bis-Tris gel systems (Invitrogen), using the SeeBlue Plus2 Pre-Stained Standard (Invitrogen) as the molecular weight marker. Western blot analysis was performed according to standard protocols [66]. Monoclonal antibodies 8B12 against Hbl L2, 2A3 and 1B8 against Hbl B, and 1C2 against NheB and Hbl L1, 1A8 against NheA (all diluted 1:15), and rabbit antiserum against NheC diluted 1:2000 [41, 67, 68] were BMS202 mouse a kind gift from Dr Erwin Märtlbauer

(Ludwig-Maximilians-Universität, Munich, Germany). For Selleckchem Rabusertib detection of CytK, rabbit antiserum diluted 1:2000 was used [24]. The Vero cell cytotoxicity assay was performed as described [35] and measures the percentage inhibition of C14-leucine incorporation in cells due to the cells being subjected to toxins, calculated relative to a negative control where cells were not subjected to toxin sample. The experiments were performed

twice, with two to four parallels in each experiment. Acknowledgements This work was supported by the Research Council of Norway (164805/I10). References 1. Stenfors Arnesen LP, Fagerlund A, Granum PE: From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev 2008, 32:579–606.PubMedCrossRef 2. Helgason E, Økstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, Hegna I, Kolstø AB: Bacillus anthracis , Bacillus cereus , and Bacillus thuringiensis – one species on the basis of genetic evidence. Appl Environ Microbiol Lck 2000, 66:2627–2630.PubMedCrossRef 3. Rivera AMG, Granum PE, Priest FG: Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis . FEMS Microbiol Lett 2000, 190:151–155.CrossRef 4. Swiecicka I, Van der Auwera GA, Mahillon J: Hemolytic and nonhemolytic enterotoxin genes are broadly distributed among Bacillus thuringiensis isolated from wild mammals. Microb Ecol 2006, 52:544–551.PubMedCrossRef 5. Gohar M, Faegri K, Perchat S, Ravnum S, Økstad OA, Gominet M, Kolstø AB, Lereclus D: The PlcR virulence regulon of Bacillus cereus . PLoS One 2008, 3:e2793.PubMedCrossRef 6.

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