Table 1. Signaling protein levels in RVFV-infected HSAECs. Protein Group I p-ERK p-STAT1 Caspase 3 cleaved Caspase 6 cleaved Caspase 7 cleaved p53 S15 p70S6 T412 Src Y416 Group II P53 S46 JNK T183/185 p90RSK S380 HSP27 MSK1 S360 p38 T180 MEK1/2 STAT3 S727 ATF2 T69/71 NF-kB S536 CREB S133 IRS S612 Src Y527 Group III AKT S473 XIAP Bim Puma LC3b PKR T451 FOX T24/31 Total protein levels ERK total 38 total JNK total AKT total Actin 48 h.p.i. Control M1 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 CI2 0.3 0.3 0.4 0.1 0.1 0.1 0.1 0.1 0.0 0.3 0.3 0.1 0.4 0.2 0.0 0.2 0.5 0.0 0.5 0.0 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.0 0.2 0.2 0.0 0.1 Low M 1.3 1.6 1.3 1.1 1.2 2.5 1.2 1.0 6.2 3.3 2.0 2.1 2.0 1.9 1.8 1.3 1.4 1.8 1.3 1.3 1.4 1.1 1.0 1.0 0.9 1.0 1.1 1.0 1.2 1.3 1.2 1.2 1.0 CI 0.3 0.1 0.1 0.0 0.1 0.2 0.1 0.1 0.1 0.3 0.2 0.3 0.2 0.2 0.0 0.2 0.5 0.1 0.3 0.0 0.0 0.1 0.1 0.0 0.2 0.1 0.1 0.0 0.1 0.2 0.2 0.1 0.1 High M 2.0 4.0 1.9 1.9 1.8 5.6 1.5 1.2 32 9.5 4.2 4.5 3.6 3.8 2.9 1.5 2.4 2.7 2.0 1.8 1.1 0.8 0.9 0.6 0.5 0.9 0.9 0.9 1.5 1.2 1.1 1.5 1.0 CI 0.3 0.3 0.4 0.2 0.4 0.3 0.1 0.1 0.4 1.4 0.8 0.5 2.2 0.2 0.1 0.2 0.8 0.3 0.3 0.0 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.0 0.1 0.3 0.1 0.1 0.1 72 h.p.i. Control M 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 CI 0.3 0.2 0.2 0.1 0.0 0.2 0.0 0.1 0.2 0.1 0.2 0.2 0.7 0.2 0.2 0.0 0.2 0.1 0.4 0.0 0.0 0.2 0.1 0.1 0.1 0.1 0.1 0.0 0.1 0.1 0.0 0.1 0.1 Low M 3.0 4.9 3.3 2.6 3.0 8.6 1.8 1.2 25 6.4 4.7 2.3 1.6 3.8 3.3 1.1 1.3 1.1 2.8 1.4 0.8 0.6 0.7 0.5 0.6 0.8 0.8 0.8 1.0 1.0 1.2 1.0 1.2 RVFV Cell Signaling CI 0.4 0.5 0.2 0.1 0.2 1.1 0.3 0.1 0.1 0.9 0.3 0.1 1.2 0.3 0.1 0.1 0.8 0.4 0.5 0.2 0.0 0.2 0.1 0.1 0.0 0.2 0.1 0.0 0.3 0.2 0.1 0.3 0.1 High M 3.0 4.7 3.5 3.0 2.9 5.6 1.3 1.4 5.9 2.3 2.9 1.8 1.5 2.2 1.4 0.8 1.0 0.6 1.3 0.8 0.5 0.5 0.5 0.3 0.3 0.5 0.6 0.8 1.4 0.7 1.0 1.4 1.2 CI 0.5 0.6 0.3 0.3 0.1 0.5 0.1 0.1 0.1 0.3 0.3 0.2 0.5 0.4 0.1 0.1 1.1 0.1 0.7 0.0 0.1 0.2 0.1 0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.2 0.1 1Arithmetic mean. 2Confidence interval, Student’s two-tail test, a = 0.05. RPMA data represent 3 independent experiments carried out in duplicate. The values are shown relative to the untreated HSAECs after normalization to the total cellular protein content at each time point. doi:10.1371/journal.pone.0013805.t001 Analysis of mitogen-activated protein kinase (MAPK) signaling MAPK (JNK, p38, ERK) pathways constitute a large kinase network that regulates a variety of physiological processes, such as cell growth, differentiation, and apoptotic cell death in response to different stimuli. We found that during RVFV infection in HSAECs the normalized levels of phosphorylation of MAPKs, including p38, JNK and ERK, were upregulated and followed specific patterns of changes (Table 1 and Fig. 5). As mentioned above, phosphorylation of ERK on T202/Y204 was steadily increased, although the total amount of ERK was relatively stable and even showed a tendency to decline at 72 h.p.i. in comparison with uninfected cells (Table 1 and Fig. 4, 5). In order to confirm activation of the ERK pathway, we tested the phosphorylation of MEK1/2 upstream of ERK1/2 with Western blotting (Fig. 4). As PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e13805