Forward [f] and reverse [r] primer pairs: CB58 [f] and CB57 [r] (icmW–CBU1651–icmX), CB59 [f] and CB60 [r] (icmV–dotA), CB718 [f] and CB716 [r] (dotA–CBU1647), CB603 [f] and CB602 [r] (dotB–CBU1646), PI3K inhibitor CB63 [f] and CB64 [r] (dotD–dotC–dotB), and CB62 [f] and CB61 [r] (icmT–icmS–dotD), were used to demonstrate transcriptional linkage. RT-PCR analysis of icmT, icmV, and icmW ORFs was performed using CB78 [f] and CB79 [r], CB70 [f] and CB71 [r], and CB40 [f] and CB41 [r], respectively. Oligonucleotide primers
(Table 1) for RT-qPCR analysis of icmX, icmW, icmV, dotA, dotB, and icmT were designed using primer3plus (Andreas Untergasser et al., 2007). The primer efficiency of all primer sets were within the efficiency window for the calculation method (Livak & Schmittgen, 2001; Schmittgen & Livak, 2008). Single-step RT-qPCR analysis using SuperScript III (Invitrogen) reverse transcriptase and the SYBR Green Master Mix Kit (Applied Biosystems) was performed on an ABI 7500 cycler. Each reaction contained 15 μL total volume and 20 ng total RNA. To calculate the relative temporal RNA
expression fold changes over the time course, to each individual gene was used, respectively. For each gene, the respective mean time-zero cycle threshold (CT) value was used as ABT-737 mouse the baseline reference point for all other (respective) mean time point CT values over the evaluation period. Therefore, for each individual gene, their relative fold expression at each time point is internally referenced to time zero RNA levels. Each time-zero point
has been referenced to themselves, resulting in a calculated fold value of 1. Statistical significance between the time points was evaluated by single-factor anova with a 95% confidence interval using ms excel 2007 (Microsoft). A P-value of <0.05 was considered significant. Recombinant C. burnetii IcmT and protein-specific antibody were the same as described previously (Morgan et al., 2010). Briefly, to ensure specificity, the rabbit sera against recombinant C. burnetii IcmT were absorbed against Vero cell lysates as well as the Escherichia coli DH5α expression strain to remove cross-reactive antibodies. This antibody was designated RαIcmT. For immunoblot analysis, purified C. burnetii NMII was pelleted and resuspended in protein lysis/running buffer [Tris-HCl, pH 6.8, 62.5 mM, much sodium dodecyl sulfate (SDS) 2%, glycerol 25%, bromophenol blue 0.01%, and 2-mercaptoethanol 5% added before loading]. Protein representing 108C. burnetii genome equivalents and 104 Vero cells, respectively, was separated by 16% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Whatman, Dassel, Germany) along with a protein ladder (Bio-Rad, Hercules, CA). Immunoblot analysis was carried out using a Pico Western Chemilluominescent Kit (Pierce, Rockford, IL) following the manufacturer’s directions using the RαIcmT primary antibody at a 1 : 1000 dilution in a hybridization buffer.