Where appropriate, we calculated the percentage of secretion as the ratio between the amounts of secreted protein (in the culture supernatant fraction) relative to the total amount of protein (in the culture supernatant and in the bacterial pellet fractions). The results from the quantifications are the average ± standard

error of the mean (SEM) from at least three independent experiments. Detailed results for each protein analyzed are in Additional file 3: Table S3. Y. enterocolitica translocation assays Analyses of protein translocation into host cells by Y. enterocolitica were done essentially as previously described [49, 50]. In brief, Y. enterocolitica strains were grown in brain heart infusion (BHI; Scharlau) medium overnight at 26°C with continuous shaking (130 rpm). Bacteria were then diluted to an optical density at 600 nm MLN2238 purchase of 0.2 in fresh BHI and cultured in the same conditions GANT61 order for 2 h. Subsequently, the yop regulon was induced by incubation for 30 min in a shaking water bath (130 rpm) at 37°C. Bacteria were then washed with DMEM supplemented

with 10% (v/v) FBS and added to HeLa 229 cells, grown overnight in 24-well plates (1×105 cells/well), by using a multiplicity of infection of 50. The infected cells were incubated at 37°C in a humidified atmosphere of 5% (v/v) CO2. After 3 h of incubation, extracellular bacteria were killed by adding gentamicin (50 μg/ml), and the cells were incubated in the same conditions for additional 2 h. The infected cells were then harvested on ice, washed with phosphate-buffered saline (PBS), ressuspended in PBS containing 0.1% (v/v) Triton X-100 and a protease inhibitor cocktail (Sigma), and incubated for 10 min on ice. The samples were centrifuged (15,000 g for 15 min at 4°C) and Triton-soluble and Triton-insoluble HeLa cell lysates were loaded on sodium dodecyl sulfate-12% (v/v) polyacrilamide gels. P-type ATPase After electrophoresis,

the gels were processed for immunoblotting using 0.2 μm pore-size nitrocellulose membranes (BioRad). Immunoblotting The following antibodies were used for immunoblotting: rat Epigenetic Reader Domain inhibitor monoclonal anti-HA (clone 3F10; Roche; used at 1:1000), mouse monoclonal anti-TEM-1 (QED Bioscience; 1:500), rabbit polyclonal anti-SycO (1:1000) [51], and mouse monoclonal anti-tubulin (clone B-5-1-2; Sigma; 1:1000). Immunoblot detection was done with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare and Jackson ImmunoResearch), Western Lightning Plus-ECL (Perkin Elmer), and a ChemiDoc XRS + system (BioRad) or exposure to Amersham Hyperfilm ECL (GE Healthcare). All quantitative analyses were done with immunoblot images obtained using ChemiDoc XRS + (BioRad). Real-time quantitative PCR The expression of the newly identified candidate T3S substrates during the developmental cycle of C. trachomatis L2/434 was estimated by determining mRNA levels at different times post-infection by real-time quantitative PCR (RT-qPCR).