Fig. 5.

Knockdown of ERK1/2 decreases EGR1 expression and viral RNA. A) Primary astrocytes were transfected with various concentrations of either negative-control (siNeg) or siRNA targeting ERK1/2 (siERK1/2). At 48 h post transfection, cell lysates were collected in blue lysis buffer and analyzed by western blot. PVDF membranes were probed for levels of ERK1/2 and β-Actin was used as a loading control. B) Cells were transfected with 100 nM siNeg or siERK1/2 siRNAs for 48 h and then infected with VEEV TC-83 (MOI 5) or mock-infected. At 18hpi, total RNA was extracted and gene expression was determined using TaqMan assays for EGR1. Fold changes were calculated relative to 18S ribosomal RNA and normalized to mock samples using the ΔΔCt method. C) RNA collected as described in panel B was used for viral genomic copy determination by RT-qPCR. Results are displayed as genomic copies in logarithmic scale. Data are expressed as the Mean ± SD (n = 3). **p-value ≤0.01, ***p-value ≤ 0.001.