RVFV Cell Signaling Figure 4. Confirmation of RPMA data by Western blot using antibodies against phosphorylated and total forms of several proteins. Western blots were performed on two independent experiments. The RVFV-infected HSAECs with MOI of 0.002 and 0.0002 at corresponding time points are indicated with a + sign. Samples 1, 2, 4, 6, 8 and 11 are from untreated cells at 0, 5, 24, 30, 48 and 72 h.p.i. and samples 3, 5, 7, 10 and 13 are RVFV- infected HSAECs with MOI of 0.002 at 5, 24, 30, 48 and 72 h.p.i. Samples 9 and 12 correspond to the cells infected with MOI of 0.0002 at 48 and 72 h.p.i. doi:10.1371/journal.pone.0013805.g004 expected, transient phosphorylation of MEK1/2 on S217/S221 at 24 h.p.i. preceded activation of ERK, which took place at 48 h.p.i. (MOI of 0.002). Downstream from activated ERK1/2 the survival signaling was propagated to p90RSK (Group II in Table 1). This kinase can activate the transcription factor CREB, which promotes cell survival through transcriptional up-regulation of anti-apoptotic Bcl-2, Bcl-XL, and Bcl-1 proteins. Increased CREB phosphorylation on S133 was observed, in agreement with phosphorylation of p90RSK (Group II in Table 1). JNK1/2 (c-Jun N-terminal kinase 1/2) pathway activity can mediate apoptosis, proliferation, or survival, depending on the stimuli and cellular conditions. Interestingly, sustained JNK1/2 activity is necessary for cellular homeostasis, whereas strong stress stimuli in non-transformed cells lead primarily to JNK-mediated apoptosis [18,19]. Total amount of JNK1/2 did not change in comparison to untreated cells (Table 1, Fig. 4). Activated form of JNK1/2 phosphorylated on T183/T185 increased in infected HSAECs at 48 h.p.i. for both MOIs. This increase was followed by a strong PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e13805