PKACs promote VISA degradation by MARCH5.

(A) Knockdown of PKACs inhibits SeV-induced VISA degradation. HEK293 cells were transfected with the indicated RNAi plasmids and selected with puromycin, then infected with SeV (MOI = 1) for the indicated times. Cell lysates were analyzed by immunoblots with the indicated antibodies. (B) PKACs promotes K48- but not K63-linked polyubiquitination of VISA. HEK293 cells were transfected with the indicated plasmids for 20 h, followed by co-immunoprecipitation and immunoblotting analysis. (C) Knockdown of PKACs inhibits SeV-induced K48-linked polyubiquitination of VISA. PKACs-RNAi stable-transduced HEK293 cells were infected with SeV (MOI = 1) for the indicated times before co-immunoprecipitation and immunoblotting analysis. (D) Mutation of T54 of VISA to alanine impairs its K48-linked polyubiquitination induced by SeV. VISA-deficient HEK293 cells reconstituted with VISA or its mutants were infected with SeV (MOI = 1) for the indicated times before co-immunoprecipitation and immunoblot analysis. (E) Effects of knockdown of AIP4, MARCH5 or RNF5 on PKACα-induced phosphorylation and degradation of VISA. HEK293 cells were transfected with the indicated RNAi plasmids and selected with puromycin, then transfected with HA-VISA and increased amounts of Flag-PKACα for 20 h before immunoblot analysis with the indicated antibodies. For the second blot from the top, the same samples were treated with λ-PPase before immunoblot analysis. (F) Effects of knockdown of PKACs on activation of the IFN-β promoter triggered by SeV and knockdown of three E3 ligases. HEK293 cells were transfected with the IFN-β promoter luciferase and the indicated RNAi plasmids for 48 h before luciferase assays were performed. (G) Effects of PKACα on the levels of VISA and its mutants. HEK293 cells were transfected with Flag-VISA or its mutants and increased amounts of Flag- PKACα plasmid for 20 h before immunoblot analysis with the indicated antibodies.