Fig 5.
The ASFV I10L protein inhibits the phosphorylation of IKKβ by reducing the K63-linked ubiquitination of NEMO. (A) The ASFV pI10L inhibits the TNF-α-triggered phosphorylation of IKKβ. HEK293T cells were transfected with either the pRK (Vec) or the pFlag-pI10L plasmid. The cells were then treated with TNF-α (20 ng/mL) for 5, 10, 20, or 40 minutes, followed by immunoblotting analysis. Densitometric analysis of protein expression level was performed with ImageJ software. (B) The ASFV pI10L inhibits the K63-linked ubiquitination of NEMO. HEK293T cells were transfected with the plasmids expressing HA-K63O, Myc-NEMO, or Flag-pI10L, followed by co-IP and immunoblotting analysis. (C) ASFVΔI10L promotes the phosphorylation of IKKβ. The primary porcine alveolar macrophages (PAMs) were left uninfected or infected with ASFVWT or ASFVΔI10L (MOI = 3) for 24 hours, after which the cells were treated or untreated with TNF-α (20 ng/mL) for 10 or 20 minutes and immunoblotting analysis was performed. Densitometric analysis of protein expression levels were performed with ImageJ software. (D) ASFVΔI10L enhances the K63-linked ubiquitination of NEMO. PAMs were left uninfected or infected with ASFVWT or ASFVΔI10L (MOI = 3) for 24 hours, and the cells were then treated or untreated with TNF-α (20 ng/mL) for 20 minutes, after which co-IP and immunoblotting analysis were performed. Densitometric analysis of protein expression levels were performed with ImageJ software. Data shown are the mean ± SD from one representative experiment performed in triplicates. ***P < 0.001 (unpaired t test).