for 5 min with hydrogen peroxide, rinsed with high-salt Tris- buffered saline (CSA Buffer, Dako) supplemented with 0.1% Tween-20, blocked with avidin block solution for 10 min, rinsed with CSA buffer, and then incubated with biotin block solution for 10 min. After another CSA buffer rinse, 5-min incubation with protein block solution was followed by air drying. the slides were incubated with either a specific primary antibody diluted in Dako Antibody Diluent or, as a control, with only Dako Antibody Diluent for 30 min. Every antibody underwent extensive validation ensuring a specific detection of the antigenic protein as a single band on the Western blot of cell lysate. The slides were washed with CSA buffer and incubated with a secondary biotinylated goat anti- rabbit IgG H1L antibody (1:7500) (Vector Labs, Burlingame, CA) for 15 min. Slides were incubated with diaminobenzidine chromo- gen diluted in Dako diaminobenzidine diluent for 5 min, washed in deionized water and imaged using a Umax PowerLook III scanner (Umax, Dallas, TX) at 600 dpi. The images were analyzed with software AlphaAse FC (Alpha Innotech). For each antibody, the average pixel intensity value for negative control (staining with second antibody only) was subtracted from the average pixel intensity value for specific antibody and then normalized by the corresponding average value of the total protein intensity. Each sample dilution curve in each experiment was fitted with a nonlinear approximation equation and statistically evaluated using GraphPad Prism ver5 software (Graphpad Software, CA). The dilution of different samples chosen within the linear response interval was used to calculate relative analyte concentrations and their 95%- confidence intervals. The RPMA experiments were repeated 3 times to ensure consistent results. Signaling proteins demonstrating differences between control and challenged cells were further tested with Western blots using 15 ml of cell lysates, 1:1000-diluted primary antibodies, and 1:10000-diluted secondary antibody (goat anti-rabbit). Proteins were transferred to a nitrocellulose membrane using iBlot Gel Transfer Device (Invitrogen). The membranes were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce), and band intensities were measured with a Molecular Imager ChemiDoc XRS System (Bio-Rad, CA). The intensities of bands were calculated relative to untreated controls after densitometry using the QuantityOne 4.6.5 program (Bio-Rad). All measurements were performed in triplicate. Data are represented as mean values with 95%-confidence intervals (two-tail t-test, a = 0.05, n = 3). MP-12 infection and inhibitor studies For Western blot analyses, HSAECs were grown in HAM’s F- 12 medium for 100% confluence in 12 well plates. Next day after confluency the cells were rinsed with PBS twice and challenged at MOIs of 0.002 and 2 at 37uC, 5% CO2 for 1 h with 400 ml RVFV MP-12 propagated and titrated using the Vero cells. After 1 h the virus was removed, cells were rinsed with PBS twice and 1 ml of HAM’s F-12 medium was added to each well. The infected cells were incubated at 37uC, 5% CO2 for 24, 48 and 72 h. After each time point the cells were rinsed with PBS twice, lysed in 250 ml of lysis buffer used in the RPMA experiments, and boiled for 10 min. For the inhibitor studies, HSAECs were seeded at 50,000 cells per well in a 96-well plate. The inhibitors (from Calbiochem) were dissolved in DMSO and added to the cells for 5 h prior to infection. A DMSO-only control was included and the final concentration of DMSO was 0.1%. Final concentrations of inhibitors were: 1 mM for p38 inhibitor SB203580, 10 mM for JNK inhibitor II and 10 mM for MEK1/2 Inhibitor III. The latter is known to specifically inhibit phosphorylation of ERK1/2. Medium was removed and cells were infected with RVFV MP-12 at MOI of 0.002 in 25 ml of medium. After one hour of infection, RVFV Cell Signaling fresh medium containing inhibitors was added to cells. Superna- tants were collected at 48 h.p.i., viral RNA extracted using Ambion’s MagMAXTM-96 Viral RNA Isolation Kit, and RNA analyzed by q-RT-PCR. RVFV q-RT-PCR assays were per- formed with a titration of purified viral RNA to establish a standard curve. The primers and probe used were originally described by Drosten et al. [1]. Assay conditions were as follows: 0.4 mM primers, 0.5 mM FAM-probe, 1 mM additional MgSO4, 0.4 ml Rox reference dye, 1.0 ml RNA UltraSenseTM Enzyme Mix, 4.0 ml RNA UltraSenseTM 56 Reaction Mix. Reactions were performed in a 20 ml volume using an ABI 7000 Real Time PCR instrument. Cycling conditions were 50uC for 15 min, 95uC for 2 min, followed by 40 cycles of 95uC for 15 s and 60uC for 30 s. Results RVFV infection has a profound influence on phosphoprotein signaling in HSAECs To study host cell signaling by protein phosphorylation during RVFV infection, we used a model of HSAECs challenged with the virulent strain of RVFV (ZH-501). We hypothesized that this model would faithfully reflect signaling induced by inhaled RVFV in the infected lung epithelium. HSAECs were grown to confluency in 6- well plates. One day later the cells were infected with the virus at MOIs of 0.0002 and 0.002. The MOIs were chosen to avoid rapid apoptosis of cells and resemble the low-dose in vivo infection by mosquito bites or inhalation. RVFV was able to cause a productive infection of HSAECs as detected by a progressive cytopathic effect upon visual examination of cell monolayer morphology at different times post infection (data not shown). Cytopathic effect (rounding up and dislodging of cells from the plastic substrate) was first observed at 72 h. Quantitative analysis of the newly-formed virus released from HSAECs into culture supernatants was performed by plaque assay (Fig. 1a). In addition, the levels of intracellular virus antigen during the period from 5 to 72 h post infection (h.p.i.) were tested by Western blot of cell lysates using antibody specific to the viral proteins Gc, Gn and NSm (Fig. 1b, c). Concentration of viral antigen reached maximum at 48 and 72 h.p.i. (for MOI of 0.002 and 0.0002, respectively). Collectively these results indicate that active infection was observed in primary lung epithelial cells at both MOIs. For phosphoproteomic analysis the cell lysates were printed onto nitrocellulose membrane slides. Each slide was then probed with one of 60 different antibodies specific against phosphorylated or total forms of signaling proteins. The antibodies were selected to monitor the molecular networks involved in host responses most likely affected by virus exposure, namely survival, apoptosis, inflammation, growth, differentiation, and immune response. This RPMA technique was extensively validated in our previous studies [16] with regard to specificity of antibodies, sensitivity, and accuracy of phosphoprotein detection in cell lysates. As an example, slides stained with antibody against total and phosphorylated form of SAP/JNK, ERK and AKT and against phosphorylated form of p- 38 and PTEN are shown in Fig. 2. All samples for untreated and RVFV-infected HSAECs from different time points were printed on the same slide. Boxed dots show untreated and RVFV-infected HSAECs at 48 and 72 h.p.i. at MOI of 0.002 (high) and MOI of 0.0002 (low). Significant changes are seen in the levels of phosphorylated forms of SAP/JNK, p38, ERK and AKT for RVFV-infected cells in comparison with untreated cells, whereas total forms of these proteins demonstrate only subtle changes. PTEN illustrates the protein which phosphorylation was not affected by RVFV infection. In order to carry out quantitative analyses of RPMA data, the average total level of cellular protein at every time point was PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e13805