For the integrin blocking assay, confluence HEp-2 cells were incubated with antibodies (10 μg/ml) against α2 (P1E6, monoclonal, Chemicon International; P17301, polyclonal, Millipore), β1 (P4G1, monoclonal, Chemicon International; P05556, polyclonal, Millipore), α2β1 selleckchem (BHA2.1, monoclonal, Chemicon International)

integrins and mouse IgG (Sigma) for 30 min before the incubation with FITC-conjugated bacteria for the adhesion assay. Electron microscopy Drops of bacterial suspension fixed with 2.5% glutaraldehyde were concentrated and placed on formvar-coated copper grids for 1 min. After removal of excess fluid by placing on filter paper, the wet residues were immediately covered with the stain for 30 sec. The grid was air-dried before examination for negative staining electron microscopy. FACS analysis Surface-detection of Scl1 in E. coli was performed by FACS analysis. Approximately 1 × 107 bacteria were incubated with mouse anti-Scl1 antibody (1:1000) for 1 hr and subsequently with https://www.selleckchem.com/products/JNJ-26481585.html FITC-conjugated

goat anti-mouse IgG (1:1000, Amersham Biosciences) for 30 min. The fluorescence of adhered bacteria was analyzed by a FACS-Scan flow cytometer (Beckton-Dickinson). Surface protein isolation Outer membrane proteins were isolated from bacteria cultures according to a protocol by Fountoulakis and Gasser [36]. Briefly, the overnight E. coli culture was pelleted and the bacteria were resuspended. After shacking and a centrifugation, the new pellet was resuspended and disrupted 3 times by sonication. To remove unbroken cells and debris, sonicated bacteria were centrifuged at 3,000 rpm and subsequently

the supernatants were centrifuged at 90,000 rpm. To solubilize the inner membrane protein, the pellet was incubated with 2 ml 2% sodium N-laung sarcosinate and subsequently the supernatants were centrifuged at 90,000 rpm. The pelleted outer membrane proteins were resuspended. OmpA expression pattern performed by western blot using anti-OmpA antibody was represented as an internal control. Recombinant protein and preparation of antibody The 1.3-kb full-length sc1l gene was cloned into plasmid pQE30 to construct plasmid pPJ10. The recombinant protein was expressed after Buspirone HCl isopropyl-β-D-thiogalactopyranoside induction. The expressed protein find more containing the His6 tag was separated in a Ni-chelated column (Amersham Biosciences) and eluted by a 0 to 50 mM imidazole gradient. The purified protein was verified by SDS-PAGE and western blot analysis with anti-His monoclonal antibody (Invitrogen). Antibody against purified rScl1 was raised in 4-week-old BALB/c mice. One hundred microgram of rScl1 was applied in the initial immunization of BALB/c mice, with succeeding injections 2 and 4 wks thereafter.