Figure 1
Evaluation of total PKR and ph-PKR in different cell lines infected with HSV-1, R2621 and ΔUL49 mutant viruses. (a) HEp-2, 293T and SH-SY5Y were infected with HSV-1, vhs-deleted (R2621) and ΔUL49 mutant viruses at MOI 10 and harvested 24 h p.i. The samples were subjected to western blot to analyse both total PKR and ph-PKR. GAPDH was used as a loading control. (b) Quantification of band intensity was determined with T.I.N.A program, the ph-PKR and PKR values were normalized to GAPDH protein levels and graphically represented using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Statistical significance was tested by one-way analysis of variance (ANOVA) (*p < 0.05, **p < 0.01, and ***p < 0.001). (c) Transient transfection of VHS and mutant VHS plasmids in 293T cells. The cells were transfected with 1 μg of plasmid DNA as described in Methods. Then, 48 h post transfection, the cells were left untreated or treated with poly I:C (0,01 μg/μl) for an additional 24 h. Western blot analysis was carried out to analyse ph-PKR, PKR, GFP and GAPDH expression following transfection with pCMS-EGFP (symbolised in the figure as pCMS), pVHS, pVHSm, pVHS/pUL48/pUL49 and pVHSm/pUL48/pUL49. (d) The quantification of band intensity of ph-PKR and PKR were normalized to GAPDH protein levels and graphically represented by GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Statistical significance was tested by one-way analysis of variance (ANOVA) (**p < 0.01, and ***p < 0.001).
