SDS-PAGE was transferred to nitrocellulose for

immunological detection. Membrane was blocked selleck products with 5% skimmed milk in TBS overnight at 4°C. Subsequently, membrane was incubated with anti-OstA polyclonal antibody [14] diluted 1:500 with 5% skimmed milk in TTBS (0.5% Tween-20) for 1 h at room temperature. Horseradish peroxidase-conjugated anti-rat IgG diluted 1:3000 with 5% skimmed milk in TTBS (0.5% Tween-20) was added and membrane was incubated for 1 h at room temperature. The membrane was washed three times with TTBS (0.5% Tween-20) between the incubation steps. Electrochemiluminescence (Amersham Biosciences, Fairfield, CT) was used for detection. RNA isolation and microarray analysis of H. pylori NTUH-S1 H. pylori NTUH-S1 was grown on Columbia blood agar plates

for 48 h and further passaged on Columbia blood agar plates or 3 μg/ml click here glutaraldehyde-containing blood agar plates for 48 h. RNA was extracted using the QIAGEN RNeasy column purification kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized according to the SuperScript™ indirect cDNA Labeling System (Invitrogen). cDNA was then purified using the S.N.A.P column purification (Invitrogen) according to the manufacturer’s instructions. Aminoallyl dUTP-labeled cDNA was resuspended in 2 × coupling buffer and labeled with either Alexa Fluor 555 or 647 according to the manufacturer’s protocol (Molecular Probes, Eugene, OR). Labeled cDNA was mixed together and purified by S.N.A.P column purification. Then, the labeled cDNA was concentrated AMP deaminase with a Microcon Selleckchem 3-deazaneplanocin A YM-30 column (Millipore, Billerica, MA). The Institute for Genomic Research (TIGR) provided a H. pylori whole-genome

microarray. It consisted of 2,572 70-mer oligonucleotides, printed in quadruplicate and representing open reading frames from H. pylori 26695 and strain J99. Labeled cDNA was resuspended in filtered hybridization buffer (50% formamide, 5 × SSC, 0.1% sodium dodecyl sulfate, 0.1 M DTT, and 0.6 μg/ml salmon sperm DNA), denatured at 95°C for 5 min, and flicked for an additional minute. It was then denatured for another 5 min. The labeled probe was applied to the pre-hybridized microarray and placed in a hybridization chamber at 42°C for 16~20 h. Microarray scanning and analysis were performed on a scanner (GenePix 4000B with GenePix Pro 5.0 software; Axon, Foster City, CA). Processed microarray data files have been deposited in the Center for Information Biology Gene Expression Database (CIBEX; http://​cibex.​nig.​ac.​jp) under accession number CBX86. Construction of imp/ostA and msbA deletion mutants The gene encoding Imp/OstA with the upstream and downstream 500 bp flanking region was amplified with the genomic DNA of wild-type NTUH-S1 by PCR. The forward primer was 5′-ATGCACTCTCCAAATTTAGA-3′, and the reverse primer was 5′-GGGGCTAGGATAGGTTCTAA-3′. It was then cloned into a pGEM-T easy vector (Promega, Madison, WI).