The supernatant was collected as IM fraction and the pellet, containing the OM, was resuspended in 20 mM Tris–HCl, pH 7.5. SDS-PAGE electrophoresis with NuPage 4-12% Bis-Tris gels (Invitrogen) and Western blot analysis were performed according to standard procedures. Opa Epoxomicin supplier proteins were detected by monoclonal antibody 4B12, kindly provided mTOR inhibitor by M. Achtman. Pili were detected by monoclonal antibody

SM1, kindly provided by M. Virji. OpaB protein was detected by polyclonal antisera against NG0070 His-tagged protein; purification of the protein and mice immunization were performed as described before. Bands were visualized with Super Signal Chemiluminescent Substrate (PIERCE). Two-dimensional gel electrophoresis and image analysis 200 μg of proteins were precipitated with 0.015% sodium deoxycholate and 48% trichloroacetic acid and dissolved in 7 M urea, 2 M thiourea, 2% CHAPS, 2% ASB14, 1% DTT, 2 mM tributylphosphine, 20 mM Tris and 2% carrier ampholyte.

Proteins were absorbed overnight onto Immobiline DryStrips (7 cm; pH-gradient 3–10 non linear) and the first dimension was run using a IPGphor Isoelectric Focusing Unit (Ge Healthcare), applying sequentially 150 V for 1 hour, 500 V for 35 min, 1000 V for 30 min, 2600 V for 10 min, 3500 V for 15 min, 4200 V for 15 min and finally 5000 V to reach 12kVh. For the second dimension, strips Pritelivir mw were equilibrated as described and proteins were separated on linear 4–12% polyacrylamide gels. Bidimensional gel was acquired with a Personal Densitometer

SI (Molecular Dynamics) and images were analyzed with the software Image Master 2D v2003.02 (Ge Healthcare). In-gel protein digestion and MALDI-TOF mass spectrometry analysis Protein spots were excised from the gels, washed with 50 mM ammonium bicarbonate/acetonitrile 50/50 (v/v) and air-dried. Dried spots were digested for 2 hours at 37°C with sequencing grade modified trypsin in 5 mM ammonium bicarbonate, loaded on a matrix Rebamipide prespotted Anchorchip (PAC 384 HCCA, Bruker-Daltonics, Bremen, Germany), air-dried and washed with 70% ethanol, 0.1% trifluoracetic acid. Mass spectra were acquired on an ultraflex MALDI TOF mass spectrometer (Bruker-Daltonics). Spectra were externally calibrated by using the combination of standards present on the PAC (Bruker-Daltonics). Monoisotopic peptide matching and protein search were performed automatically by MASCOT software. Cell culture Ectocervical and Endocervical cells (Ect1/E6E7 and End1/E6E7 from ATCC) were maintained in keratinocyte serum-free medium (KSFM, Gibco) supplemented with 50 μg/mL bovine pituitary extract, 0.1 ng/mL epidermal growth factor, 0.4 mM CaCl2 and antibiotics at 37°C in 5% CO2. Transformed urethral epithelial cells (kindly provided by M.