Figure 2.

ASFV A528R inhibits the NF-κB p65 phosphorylation downstream TLR8 signaling. (A) 293T cells grown in 12-well plate (3 × 10⁵ cells/well) were transfected with A528R plasmid (0.25 µg, 0.5 µg), pTLR8 (0.5 µg) as indicated. Twenty-four hours post transfection, cells were stimulated with R848 (5 µg/mL) for 12 h, the expressions of p-p65, p65, A528R, and pTLR8 were analyzed by Western blotting. (B–D) 293T cells were transfected with A528R (0.25 µg, 0.5 µg) or vector (EV) control (0.5 µg), together with MyD88 (0.5 µg) (B), IKK-β (0.5 µg) (C), p65 (0.5 µg) (D). Twenty-four hours later, the expressions of p-p65, p65, A528R, MyD88, IKK-β, p65 were analyzed by Western blotting. (E) 293T cells grown in 12-well plate (3 × 10⁵ cells/well) were transfected with A528R (0.25 µg, 0.5 µg), pTLR8 (0.5 µg). Twenty-four hours post transfection, cells were stimulated with R848 (5 µg/mL) for 12 h. Cell lysates were fractionated into cytoplasmic and nuclear extracts, and the protein levels of p65 and p-p65 in different fractions were analyzed by Western blotting. The histone-H3 and GAPDH were also shown as the nuclear and cytoplasmic markers, respectively.