3 ± 1.8 45.5 ± 1.9 1.8 <0.0001 17.51 ± 0.81 16.64 ± 0.23 1.81 this website 0.0174 pRG198 76.9 ± 1.7 35.7 ± 1.6 2.2 <0.0001 17.48 ± 0.08 16.27 ± 0.06 2.24 0.0013 #volume of the unoccupied space available under the signal is quantitated *p-value of ≤ 0.05 is significant EMSA analysis of upstream sequences of p28-Omp14 and p28-Omp19 promoters Electrophoretic Selleck Akt inhibitor mobility shift assay (EMSA) experiments utilizing the complete promoter regions of the p28-Omp14 and p28-Omp19 of E. chaffeensis showed promoter-specific

binding of tick cell- or macrophage-derived E. chaffeensis proteins (not shown). Addition of 50 ng of specific competitor DNAs consisting of unlabeled full length promoter DNA of p28-Omp14 or p28-Omp19 abolished the shift of DNA-protein complex migration for both promoter regions. To further assess the interactions of Ehrlichia proteins with putative upstream sequences, five biotin-labelled short upstream DNA segments of p28-Omp14 (probes LY3039478 P1 to P5) (Figure 8A) and two DNA segments of p28-Omp19 (P6 and P7) (Figure 8B) promoters

were prepared and used in the EMSA experiments. The promoter sequences of genes 14 and 19 included direct repeats and palindromic sequences [25]. The probes included one or more of the sequences. Three of the five probes for the p28-Omp14 promoter region exhibited significant shift in mobility in the presence of protein lysate from macrophage derived E. chaffeensis compared to the controls which contained probe alone with no lysate added or when non-specific protein was added to the probe fragments (Figure 9A). A shift in mobility was also noted in the interaction with one probe segment of the p28-Omp19 promoter region when

the protein lysate was added (Figure 9B). Addition of a 50-fold excess of unlabeled specific-competitors in the binding reactions significantly reduced the mobility shift of the probes. Densitometry analysis of the mobility shifted fragments differed for each probe compared to the non-shifted fragments. The P1 probe had 84% shift which reduced to 29% when competitor DNA was added; P2 and P3 probes had about 31%, and 27% shifts, respectively, and the shifts for these probes were completely Amobarbital abolished in the presence of specific competitors. The p28-Omp19 promoter region probe had about 23% shift which was reduced to 10% in the presence of specific competitor. Figure 8 Sequences of EMSA probes used in this study. Sequences of p28-Omp14 P1-P5 (panel A) and p28-Omp19 P6 and P7 (panel B) represent promoter segments utilized in the EMSA experiments. Figure 9 EMSA using short segments of three biotin-labeled probes of p28-Omp14 (panel A) and one p28-Omp19 (panel B) promoter segments. Addition of E.