Expression and purity of the fusion protein was determined by SDS-PAGE according to standard protocols [45]. Immunoblot analysis was performed as described by Ausubel et al. (1996) using anti-AatA antibody (see below). Antibody production The anti-AatA antibody was produced

in New Zealand White rabbits as follows: 300 μg highly purified fusion protein solved in PBS were mixed with an equal volume of adjuvant ISA 206 (SEPPIC S.A., Puteaux, France) and subcutaneously injected into the back of the rabbits at seven different sites. Selleckchem Proteasome inhibitor immunization was repeated thrice at 2-week intervals. Ten days after the final immunization blood was collected by cardiac puncture under terminal anaesthesia, and serum samples were prepared and frozen at -20°C. find more Quantitative real-time PCR Overnight cultures of E. coli were diluted to an Adavosertib order OD600 = 0.1 in fresh LB. Bacteria were grown to the logarithmic phase (OD600 = 0.8), harvested, and cell pellets were resuspended in Trizol (Invitrogen GmbH, Karlsruhe, Germany). Total RNA was isolated according to the manufacturer’s protocol followed by digestion of the genomic DNA using RQ1 RNase-Free DNase (Promega, Mannheim, Germany). cDNA synthesis was then performed using random hexamere-primers and the MMLV reverse transcriptase

following the manufacturer’s protocol. cDNA aliquots corresponding to 150 ng of total RNA were semi-quantitatively analyzed using sense (aatA RT-F) and antisense oligonucleotides (aatA RT-R) of the target gene aatA and analyzed by real-time PCR (Applied Biosystems StepOne) with the SYBR® Green method. The relative gene expression

of aatA was normalized to the expression of the housekeeping gene gyrB, which was amplified using primers 4057 and 2521 (Additional file 1: Table S1), via the ΔΔCt method. PCR efficiency (> 90%) for each of the gene was checked via standard dilution curves. Immunoblot For immunoblot experiments, overnight cultures of E. coli were diluted 1:100 into fresh LB. The bacteria were grown to the logarithmic phase, harvested, resuspended in protein denaturation buffer and boiled for 10 min [48]. Total protein extracts were loaded on 10% SDS gels and transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia new Biotech, Shanghai, China) using a semi-dry blotting apparatus (TE77, Amersham Pharmacia Biotech) and a buffer containing 39 mM glycine, 48 mM Tris base, 20% methanol, and 0.037% SDS. Serum raised against the passenger domain of AatA was used as primary antibody and horseradish peroxidase-conjugated antirabbit immunoglobulin as secondary antibody. Tetra methyl benzidine was used as the substrate to visualize protein bands. Adherence assay For adhesion studies, the IMT5155 aatA ORF and the 99 bp upstream containing the putative native aatA promoter were amplified and cloned into pMD18T (TaKaRa, Dalian, China) vector using oligonucleotides WSH18F and WSH16R adding the restriction enzyme recognition sites BamHI and HindIII.