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. 2017 Aug 24;91(18):e00880-17. doi: 10.1128/JVI.00880-17

FIG 7.

FIG 7

NAP1L1 controls RELA levels and IRF3 activation. (A) NAP1L1 depletion affects RELA levels and IRF3 phosphorylation. U2OS cells were transduced with LV for shNAP1L1 or shCTRL and subsequently transfected with 1 μg poly(I·C) using Lipofectamine (lipo). Protein levels as indicated were monitored by WB at 1, 2, and 4 h posttransfection of poly(I·C). (B) NAP1L1 depletion decreases RELA protein levels. Blots as in panel A were quantified to measure RELA and IRF3 protein levels using ImageJ. Shown is the shNAP1L1/shCTRL ratio in cells not transfected with poly(I·C). Averages for 3 independent replicates are shown with standard deviations. (C) NAP1L1 depletion affects IRF3 phosphorylation. Blots as in panel A were quantified to measure RELA and IRF3 phosphorylation levels using ImageJ. Shown is the ratio of phosphorylated protein to total protein in cells transfected with poly(I·C). Averages for 3 independent replicates are shown with standard deviations. (D) NAP1L1 depletion reduces RELA nuclear translocation. U2OS cells were transduced with LV for shNAP1L1 or shCTRL and subsequently transfected with 1 μg poly(I·C) for 8 h. Cells were then fixed and stained for RELA. (E) NAP1L1 depletion reduces RELA nuclear translocation. Around 500 cells from the experiment for panel D were counted for each condition to calculate the percentage of RELA nuclear translocation. Averages for 3 independent replicates are shown with standard deviations. (F) NAP1L1 depletion reduces IRF3 nuclear translocation. U2OS cells were transduced with LV for shNAP1L1 or shCTRL and subsequently transfected with 1 μg poly(I·C) for 8 h. Cells were then fixed and stained for IRF3. (G) NAP1L1 depletion reduces IRF3 nuclear translocation. Around 500 cells from the experiment for panel F were counted for each condition to calculate the percentage of IRF3 nuclear translocation. Averages for 3 independent replicates are shown with standard deviations. (H) HCV NS5A inhibits TBK1-mediated activation of IFN-β. HEK 293T cells were transfected with expression vectors for FLAG-tagged TBK1, NS5A, or the NS5A-m2 mutant together with a reporter plasmid carrying the firefly luciferase (Fluc) gene under the control of the IFN-β promoter (pIFNβ-Luc) and the control pCMV-Renilla. Relative light units (RLUs) of luciferase activity were measured in quintuplicate independent experiments, normalized for Renilla, and represented as fold change over mock treatment ± SD.