5 ml of lysis buffer (150 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM Magnesium Acetate, 1 mM EDTA, 2 mM DTT and 10% glycerol) and the pellet was resuspended in 1 ml of lysis

buffer containing protease inhibitors Adriamycin in vitro (Roche Diagnostic Labs, Indianapolis, IN). The cell suspension was sonicated four times at 8.5 setting, 30 sec each time to lyse E. chaffeensis organisms. The cell lysates were centrifuged at 15,560 × g for 15 min at 4°C to pellet the insoluble fraction and the supernatant containing soluble proteins of E. chaffeensis was collected into PU-H71 supplier sterile micro centrifuge tubes as 25 μl aliquots containing protease inhibitor mix and stored at -80°C until use. Protein concentration of the protein lysates, prior to adding the protease inhibitor mix, was estimated as described above. Electrophoretic mobility shift assay (EMSA) DNA sequence segments spanning one or more putative regulatory sequences of p28-Omp14 or p28-Omp19 gene promoters

were amplified from E. chaffeensis Arkansas isolate genomic DNA using sequence specific primers and 5′end biotin-labeled reverse primers (Table 1) and evaluated for their interaction with the protein lysates. EMSA experiments and detection were carried out according to established protocols [57, 58] with a radioactive nucleotide incorporated DNA probes or using the LightShift Chemiluminescent EMSA kit (Pierce Biotechnology, Rockford, Illinois, USA) according to the specifications of the manufacturer. The assay mixtures included a non-specific DNA (salmon sperm DNA or poly dI.dC at a high concentration of 240 μg/ml or 50 μg/ml, respectively) to eliminate non-specific interactions. Briefly, about buy VX-680 1 ng of each of the full length or biotin-labeled partial upstream sequences was used

in each reaction together with 5 μg of the E. chaffeensis whole-cell protein lysate. About 50 ng of unlabeled specific probe sequences were used as competitors. Bovine serum albumin (BSA) was included in each experiment as a non-specific protein control. The protein concentration in E. chaffeensis protein lysates used in these experiments was similar to the work reported earlier [41, 49, 58]. Statistical analysis We carried out two-tailed t-tests with equal variances for densitometry analysis and unequal variances for the real-time RT-PCR analysis to comparatively analyse the effect of addition of E. chaffeensis whole cell protein lysate on transcription of p28-Omp14 (pRG147) check and p28-Omp19 (pRG198) promoters. Acknowledgements This work is supported by National Institutes of Health grant AI070908. We thank Dr. Ming Tan of the University of California, Irvine, CA for providing the G-less cassette parent plasmid, pMT504. We also acknowledge Chuanmin Cheng for her technical assistance. This manuscript is a contribution from the Kansas Agricultural Experiment Station, number 11-283-J. References 1. Chen SM, Dumler JS, Bakken JS, Walker DH: Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease.