Fig. 6.

Knockdown of PERK decreases EGR1 expression and viral RNA. A) Primary astrocytes were transfected with various concentrations of a negative-control (siNeg) or siRNA targeting PERK (siPERK). At 48 h post transfection, cell lysates were collected using blue lysis buffer and analyzed by western blot. PVDF membranes were probed for levels of ERK1/2 and β-Actin as a loading control. B) Cells were transfected with 100 nM siNeg or siPERK siRNAs for 48 h and then infected with VEEV-TC83 (MOI 5) or mock-infected. At 18hpi, total RNA was extracted and gene expression was determined using TaqMan assays for EGR1 gene. 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 ≤ 0.0001.