Following three washing steps with PBS, the cells were permeabilized with buffer A (50 mM EDTA, 20 mM Luminespib order Tris-HCl, 1.8 g/l glucose, pH 8.0) containing freshly added 0.1% Triton X-100 for 5 min at RT. Buffer A was replaced by three washing steps with

10058-F4 molecular weight buffer B (10 mM EDTA, 25 mM Tris-HCl, 1.8 g/l glucose, pH 8.0) and buffer B plus 5 g/l lysozyme for staining of proteins in the bacterial cytosol or without lysozyme for staining of intracellular secreted proteins was added for 1 h at 4°C. Cells were washed again with PBS and incubated for 1 h at RT in blocking solution (10% goat serum, 1% bovine serum albumin, 4% sucrose in PBS). SseB was stained using polyclonal antisera against recombinant SseB from rabbit [7] and anti-rabbit Alexa488 (Molecular Probes, Invitrogen). S. Typhimurium was stained with rabbit anti-Salmonella O1,4,5,12,27 antiserum (Difco) conjugated with DyLight 547 NHS ester (Pierce). The lysosome-associated membrane protein 1 (LAMP-1) that is associated with SCV in infected cells was stained using a monoclonal antibody H4A3 from rat (1:100, developed by J.T. August, J.E.K. Hildreth, was obtained from the Developmental Studies Hybridoma Bank developed

under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242) and anti rat Cy5 (1:500, Jackson). All antibodies were diluted in blocking solution. Following immuno-staining, the coverslips were mounted on Fluoprep (bioMèrieux) and sealed with Entellan (Merck). Images www.selleck.co.jp/products/AG-014699.html of the samples were recorded using a Alvocidib cell line confocal laser-scanning microscope (Leica TCS-NT). Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft grant HE1962/8-3. S.U.H. was a fellow the graduate school BIGSS ‘Lead structures

of cell function’ of the Elite Network Bavaria. We like to thank Daniela Jäckel for excellent technical support of the work. Electronic supplementary material Additional file 1: Effect of various deletions in sseD on synthesis and secretion of SseD in vitro. S. Typhimurium WT or ΔsseD without plasmid, harboring plasmid psseD for complementation of the sseD deletion, or plasmids for the expression of various sseD mutant alleles (psseDΔx) were grown in 400 ml minimal medium PCN-P (0.4 mM) at pH 5.8 to induce SPI2 expression as well as protein secretion by the SPI2-T3SS. For analyses of protein synthesis, equal amounts of bacterial cells as adjusted by OD600 were harvested and resuspended in SDS-PAGE sample buffer (total cell fraction). Secreted protein bound to the bacterial surface was released by mechanical shearing and precipitated from bacteria-free supernatant (detached fraction) and secreted proteins in the supernatant were precipitated by addition of 10% TCA (final concentration).