Ac N.A [45]    pKD3 Red Recombinase template plasmid (CmR) N.A N.A [45]    pKD4 Red Recombinase template plasmid (KanR) N.A N.A [45]    pTrc99A High-copy number expression vector (AmpR) N.A N.A [49]    pFliC Derivative of pTrc99A encoding fliC from EPEC E2348/69 (AmpR) N.A N.A This study    pFliCEscF Derivative of pTrc99A encoding fliC and escF from EPEC E2348/69 (AmpR) N.A N.A This study    pCDNA3 Eukaryotic expression vector N.A N.A Promega aKan, kanamycin; Cm, chloramphenicol; Amp, ampicillin. bFAS, Fluorescent actin staining test. cN.A., not applicable. Isolation of secreted proteins

EPEC was inoculated into 5 ml of LB and grown overnight at 37°C with shaking. EPEC was routinely diluted 1:100 in DMEM containing 44 mM NaHCO3 buffered with 25

mM HEPES and grown at 37°C with shaking. Bacterial supernatants were analyzed at selleck compound mid- to late-log phases of growth [42]. To ensure removal of bacteria and cellular debris, the bacterial supernatants were filtered through 0.45 μm pore filters (Millipore, Bedford, MA) [43]. The cell-free supernatants were precipitated with a final 10% w/v trichloroacetic acid (TCA) solution and subsequent centrifugation at 13,000 rpm for 45 min at 4°C followed by three methanol washes. Equal amounts selleck screening library of proteins were analyzed by SDS-PAGE and by two-dimensional gel electrophoresis. Proteins of interest were subjected to mass spectrometry. SDS-PAGE and immunoblotting The bacterial suspensions were adjusted to an absorbance of 1.0 at OD600. Equal numbers of bacteria

were used to prepare whole cell extracts in sample denaturation buffer and separated by 12% SDS-PAGE. The gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad, Hercules, CA) or transferred onto nitrocellulose membranes (Pall Life Science, Pensacola, FL) for immunoblotting. The immobilized proteins were incubated with primary antibodies against H6 flagellin (Statens Serum Institut, Denmark) or cytoplasmic protein DnaK (Assay Designs, Ann Arbor, MI) followed by incubation with goat anti-rabbit (Sigma, St. Louis, MO) or sheep anti-mouse IgG (Chemicon, Melbourne, Australia) conjugated much to alkaline-phosphatase. Antibody binding was detected with chemiluminescent reagent (Astral Scientific, Gymea, NSW, Australia). Two-dimensional Gel Electrophoresis Proteins secreted from approximately 109 cells (~120 μg) were precipitated with a final 10% w/v TCA solution and material was resuspended in 460 μl of following sample solution: 5 M urea (Amersham Pharmacia Biotech, Sweden), 2 mM tributylphosphine (TBP) 2% CHAPS, 2% (v/v) carrier ampholytes (Bio-Rad, CA, USA), 2% SB 3–10 or 2% SB 4–7 and trace of bromophenol blue (Pharmacia Biotech) by vortexing [44]. Insoluble material was removed by centrifugation at 12 000 × g for 10 min. The 460 μl samples were used to passively rehydrate pH 3–10 or pH 4–7 immobilized pH gradient dry strips for 18 h at room temperature (Bio-Rad).