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. 2017 Jul 27;7:6737. doi: 10.1038/s41598-017-06934-1

Figure 4.

Figure 4

TRAF6 promotes NF-κB activation and increases IFN-β and IL-6 expression in PAMs. (a) TRAF6 overexpression promotes NF-κB activity. Stable cell lines were co-transfected with pNF-κB-luc and pRL-TK plasmids. At 12 hpt, the cells were mock-infected, CSFV-infected, or poly (I:C)-stimulated for an additional 12 h. The dual-luciferase reporter assay determined NF-κB luciferase activity. (b) Western blot for p65, p-p65, and TRAF6 expression in mock- and CSFV-infected CMV-TRAF6 cells. Cells stably expressing CMV or CMV-TRAF6 were mock- or CSFV-infected for 24 h, and the cell lysate was subjected to Western blot. (c) TRAF6 knockdown inhibits NF-κB activity initiated by poly (I:C). Stable shN and TRAF6-sh2 cells were treated with poly (I:C), with shN cells infected with CSFV for 24 h, and PAMs treated with poly (I:C) as a mock condition. Cell lysates were used to determine NF-κB luciferase activity. (d) Western blot for p65, p-p65, and TRAF6 expression in TRAF6-sh2 cells. Stable shN and TRAF6-sh2 cells were treated with poly (I:C), with shN cells infected with CSFV for 24 h, PAMs treated with poly (I:C) as a mock condition. Cell lysate was subjected to Western blot. The relative levels of p-p65 protein were estimated by histograms representing density readings of the gel bands, and the ratios were calculated relative to the β-actin control. (e) TRAF6 overexpression increases IFN-β and IL-6 expression in PAMs. Cells stably expressing CMV or CMV-TRAF6 were mock- or CSFV-infected for 24 h, followed by extraction of total RNA to determine relative IFN-β and IL-6 mRNA expression. The cell culture supernatant was used to determine IFN-β and IL-6 protein levels. (f) TRAF6 knockdown decreases IFN-β and IL-6 expression in PAMs. The stable shN and TRAF6-sh2 cells were mock- or CSFV-infected for 24 h. Relative IFN-β and IL-6 mRNA expression and protein levels were determined as described. The data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the controls calculated using Student’s t-test.