PKACs phosphorylate VISA at T54.

(A) Effects of PKACs and their kinase inactive mutants on VISA modification. HEK293 cells were transfected with HA-VISA and increased amounts of the indicated expression plasmids for 20 h before immunoblot analysis with the indicated antibodies. (B) VISA is serine/threonine phosphorylated by PKACs. HEK293 cells were transfected with HA-VISA and the indicated expression plasmids for 20 h. Cell lysates were immunoprecipitated with anti-HA, and the immunoprecipitates were treated with buffer or lambda protein phosphatase (λ-PPase) and then analyzed by immunoblots with the indicated antibodies. (C) Effects of PKACα on VISA and its mutants. HEK293 cells were transfected with the indicated expression plasmids for 20 h and then analyzed by immunoblots with the indicated antibodies. (D) Effects of VISA and its mutants on IFN-β promoter activation. HEK293 cells were transfected with the IFN-β promoter luciferase and Flag-VISA or its mutants plasmids for 20 h before luciferase assays were performed. The blot shows the expression levels of the transfected VISA and its mutants. (E) Effects of VISA and its mutants on SeV-induced transcription of IFNB1 gene. VISA-deficient HEK293 cells reconstituted with VISA or its mutants were infected with SeV (MOI = 1) for the indicated times before qPCR analysis. (F) Effects of VISA and its mutants on SeV-induced TBK1 and IRF3 phosphorylation. VISA-deficient HEK293 cells reconstituted with VISA or its mutants were infected with SeV (MOI = 1) for the indicated times before immunoblot analysis with the indicated antibodies. (G) PKACs phosphorylate VISA at T54. HEK293 cells were transfected with HA-VISA, Flag-tagged PKACα and Flag-PKACβ and their mutants for 20 h. Cell lysates were analyzed by immunoblots with the indicated antibodies. (H) Endogenous VISA is phosphorylated at T54 by PKACs after SeV infection. HEK293 cells were transfected with the indicated plasmids and selected with puromycin, then infected with SeV (MOI = 1) for the indicated times. Cell lysates were subjected to immunoprecipitation and immunoblot analysis with the indicated antibodies. (I) Effects of PKACs knockdown on activation of the IFN-β promoter by VISA and its mutants. HEK293 cells were transfected with the IFN-β promoter luciferase and PKACs RNAi plasmid for 36 h, then transfected with Flag-VISA or its mutants for 20 h before luciferase assays were performed.