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. 2021 Apr 28;11:9188. doi: 10.1038/s41598-021-88610-z

Figure 5.

Figure 5

Combination of S6D and S97D phosphomimetic mutations prevents PKR activation by poly(I:C). PKR-KO cells were transfected either with wt PKR or Ser6- and/or Ser97-to-Asp PKR mutants for 40 h. Cells were next transfected with poly(I:C) for 8 h and then lysed for western blot analysis. The FB09 sample (HeLa cells infected with LM60V-mutant TMEV for 12 h) was used as positive control for PKR activation (a) Western blot detection of PKR, PKR-pT446, eIF2α, eIF2α-pS51, cleaved PARP and β-actin. (b) Quantification of the PKR-pT446 / PKR ratio. (mean ± SEM, n = 4). (c) Quantification of apoptosis by the cleaved PARP / β-actin ratio. (mean ± SEM, n = 4). 1-way ANOVA was used for multiple comparisons between poly(I:C)-treated samples. Western blots were quantified by chemiluminescence using a CCD camera (Fusion Solo-S, Vilber) and quantified within the limits of the dynamic range, using software Bio1D version 15.08. Graphs were generated with Excel 2011 (Microsoft). Statistical analysis was performed using Prism7 (Graphpad).