PKACs impair VISA aggregation.

(A) Oligomerization of VISA and its mutants. HEK293 cells were transfected with the indicated expression plasmids for 20 h before co-immunoprecipitation and immunoblot analysis with the indicated antibodies. (B) Effects of T54 mutation on SeV-induced aggregation of VISA in the mitochondria. Crude mitochondrial extracts were prepared from HEK293 cells reconstituted with VISA and its mutants and infected with SeV (MOI = 1) for the indicated times. The extracts were fractionated by SDD-AGE and SDS-PAGE and analyzed by immunoblots with the indicated antibodies. (C) Effects of PKACs knockdown on SeV-induced VISA aggregation. Crude mitochondrial extracts were prepared from control or PKACs-RNAi stable-transduced HEK293 cells infected with SeV (MOI = 1) for the indicated times. The extracts were fractionated by SDD-AGE and SDS-PAGE and analyzed by immunoblots with the indicated antibodies. (D) Effects of T54 mutation on SeV-induced aggregation of VISA in MEF cells. Crude mitochondrial extracts were prepared from MEF cells reconstituted with VISA and its mutants and infected with SeV (MOI = 1) for the indicated times. The extracts were fractionated by SDD-AGE and SDS-PAGE and analyzed by immunoblots with the indicated antibodies. (E) Confocal microscopy for virus-induced aggregation of VISA and its mutants. Flag-tagged VISA or its mutants were reconstituted into Visa^-/- MEFs. The cells were infected with SeV (MOI = 1) for the indicated times before confocal microscopy. The images are representative of > 50% of the cells under examination.