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. 2021 May 12;13(5):892. doi: 10.3390/v13050892

Figure 2.

Figure 2

siRNA knockdown of PERK has no effect on VEEV replication in U87MG or 293T cells. U87MG cells were transfected with 100 nM of siNeg or siPERK siRNAs. At 48 h post transfection, cells were mock infected or infected with VEEV TC-83 (MOI 5). (A) Viral replication was analyzed via plaque assays using supernatants collected from U87MG cells at 16 hpi. N = 3, ns = not significant. (B) Cell lysates were collected at 16 hpi and analyzed by immunoblot. PVDF membranes were probed for levels of PERK, VEEV GP, and VEEV capsid. β-Actin was used as a loading control. (C,D) Data show the quantitation of the respective immunoblots. Protein levels expressed in each blot were normalized to β-actin and normalized values were calculated relative to siNeg-transfected cells. N = 3, *** p ≤ 0.001. (E) 293T cells were transfected with 100 nM of siNeg or siPERK siRNAs. At 48 h post transfection, cell lysates were collected and analyzed by immunoblot. PVDF membranes were probed for levels of PERK. β-Actin was used as a loading control. (F) 293T cells were transfected with 100 nM of siNeg or siPERK siRNAs. At 48 h post transfection, cells were infected with VEEV TC-83 (MOI 5). Viral replication was analyzed via plaque assays using supernatants collected at 18 hpi. N = 3, ns = not significant.