PKACs target VISA in the virus-triggered pathways.

(A) VISA interacts with PKACα and PKACβ. HEK293 cells were transfected with the indicated plasmids for 20 h before co-immunoprecipitation and immunoblot analysis with the indicated antibodies. (B) Distribution of PKACs. HEK293 cells were infected with SeV (MOI = 1) for the indicated times and then fractionated the subcellular fractions were equilibrated to equal volumes and analyzed by immunoblots with the indicated antibodies. (C) Endogenous VISA is associated with PKACs in the mitochondria. HEK293 cells were infected with SeV (MOI = 1) for the indicated times. The mitochondria were isolated by cell fractionation and the mitochondrial lysates were subjected to immunoprecipitation and immunoblot analysis with the indicated antibodies. (D) Effects of PKACs knockdown on ISRE activation by various components. HEK293 cells were transfected with ISRE luciferase and PKACs RNAi plasmids for 36 h, then transfected with the indicated expression plasmids for 20 h before luciferase assays were performed. (E) Effects of H89 on PKACs-mediated inhibition of VISA activity. HEK293 cells were transfected with the IFN-β promoter luciferase and the indicated expression plasmids for 20 h, then transfected with VISA plasmid for 20 h and treated with H89 (10 μg/mL) or left untreated for 8 h before luciferase assays were performed.