gingivalis cultures were centrifuged for 30 min at 20,000 × g at 4°C and the supernatants were filtered through a 0.22-μm pore-size filter (Roth). Bacterial pellets were washed with PBS and suspended in PBS to OD660 = 0.1. To separate outer-membrane vesicles, the filtered culture medium was centrifuged for 2 h at 100,000 × g. For HmuY expression analysis, samples corresponding to 5 μl of the bacterial culture at OD660 = 0.1 or 20 μl of the culture medium were separated by 15% SDS-PAGE and transferred onto nitrocellulose membranes (Schleicher & Schuell). Nonspecific binding sites were blocked with 5% skim milk in PBS. HmuY was visualized with polyclonal anti-HmuY rabbit

serum (Lampire) and secondary goat anti-rabbit IgG antibodies conjugated with horseradish peroxidase (HRP; Sigma), both used at 1:10,000 dilutions. The reaction was developed using chemiluminescence reagents (Western Lightning Plus-ECL; Perkin Elmer). To determine P. gingivalis mTOR inhibitor autolysis, the presence of Fur was examined in both cells

and culture medium using Western blotting with rabbit polyclonal antibodies raised against the synthetic peptide derived from the amino-acid sequence of Fur (CILADKDLRPPRFSY; GeneScript). Enzyme-Linked Immunosorbent Assay (ELISA) Epacadostat in vitro Levels of anti-HmuY antibodies in rabbit find more sera were determined by ELISA. For this purpose, 96-well polystyrene plates (Polysorp; Nunc) were coated for 1 h at 37°C with 100 μl/well HmuY in PBS. The plates were washed three times with 200 μl of PBS prior to blocking for 1 h at 37°C with 200 μl of 2% bovine serum albumin (BSA) dissolved in PBS and then washed three times with 200 μl of PBS. Two-fold serum dilutions or 1:10,000 serum dilutions (100 μl of pre-immune, test I, test II, and immune

serum) were prepared in PBS and incubated for 1 h at 37°C. After washing, antibody binding was detected using goat anti-rabbit IgG conjugated with HRP. After three final washes, a substrate solution (100 μl) containing 0.05% o-phenylenediamine (Sigma) with 0.01% H2O2 was added for color development at room temperature. The reaction was stopped after 15 min by adding 25 μl of 12.5% H2SO4 and the absorbance was measured at 450 nm using a Multiskan Ascent microplate reader (Thermo Electron Corporation). Whole-cell ELISA, dot-blotting, and FACS analyses As an additional method of HmuY detection, cell surface www.selleck.co.jp/products/Staurosporine.html staining with anti-HmuY antibodies was performed using whole-cell ELISA, dot-blotting, and flow cytometry (FACS) analyses. P. gingivalis cells grown to OD660 = 1.0 were used for these experiments. For the ELISA and dot-blotting analyses, washed cells at several dilutions were adsorbed on the surface of microtiter plates or nitrocellulose membranes. Nonspecific binding of antibodies was prevented by incubation with 1% bovine serum albumin and 2% bovine fetal serum (Sigma) before the addition of rabbit pre-immune or anti-HmuY immune serum (1:10,000) or purified IgG fractions (100 ng/ml).