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. 2018 Sep 7;8:13418. doi: 10.1038/s41598-018-31734-6

Figure 4.

Figure 4

USP4 positively regulates the antiviral NF-κB pathway. (A) Western blot analysis of RD cells transiently transfected with control plasmid or HA-USP4 plasmid for 48 h, followed by treatment with EV71 for the indicated time. The cell lysates were analysed by immunoblotting using anti-pIRF3, anti-IRF3, anti-HA, and anti-GAPDH antibodies, respectively. (B) Western blot analysis of RD cells transiently transfected with control plasmid or HA-USP4 plasmid for 48 h, followed by treatment with EV71 for the indicated time. The cell lysates were analysed by immunoblotting using anti-pNF-κB P65, anti-NF-κB P65, anti-TRAF6, anti-HA, and anti- GAPDH antibodies, respectively. (C) Western blot analysis of HEK293T cells transfected with control plasmid or HA-USP4 plasmid for 48 h, followed by treatment with poly (I:C) (HMW; 30 μg/ml) for various lengths of time. The cell lysates were analysed by immunoblotting using anti-pNF-κB P65, anti-NF-κB P65, anti-TRAF6, anti-HA, and anti- GAPDH antibodies, respectively. (D) Luciferase activity in HEK293T cells transfected with a luciferase reporter for NF-κB, together with either a control plasmid or HA-USP4 expression plasmid, followed by either no treatment or treatment with intracellular HMW poly (I:C) (1 μg/ml). (E,F) q-PCR and western blot analysis of HEK293T cells transfected with control siRNA (Ctrl siRNA) or USP4-specific siRNA 1, siRNA 2 for 48 h. (G) Luciferase activity in HEK293T cells transfected with a luciferase reporter for NF-κB, together with either control siRNA or USP4-specific siRNA 2, followed by either no treatment or treatment with intracellular HMW poly (I:C) (1 μg/ml). Data in (B–D,G) are presented as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. The independent experiments were performed in triplicate.