Figure 5.

BAG6 regulates the aggregation and recruitment of downstream signaling molecules of VISA. (A) Overexpression of BAG6 inhibits SeV-induced aggregation of VISA. HEK 293 cells (2×10⁶) were transfected with vector or Flag-BAG6 for 20 h, and cells were infected with SeV (MOI = 1) for 8 h before harvest. Then, the lysates were fractionated by SDD-AGE and SDS-PAGE before immunoblotting analysis. (B) BAG6-deficiency increases SeV-induced aggregation of VISA in NIH3T3 cells. Wild type and BAG6-deficient NIH3T3 cells were untreated or treated with SeV (MOI = 1) for 8 h before harvest. Then, the lysates were fractionated by SDD-AGE and SDS-PAGE before immunoblotting analysis. (C) BAG6-deficiency increases SeV-induced aggregation of VISA in BMDMs. Wild-type BMDMs were infected with lentivirus containing BAG6-CRISPR Cas9 sgRNA for 48 h before viral infection. Wild type and BAG6-deficient BMDMs cells were untreated or treated with SeV (MOI = 1) for 8 h before harvest. Then, the lysates were fractionated by SDD-AGE and SDS-PAGE before immunoblotting analysis. (D–G) Overexpression of BAG6 inhibits the interaction of VISA and TRAF2 specifically. HEK 293 cells (2×10⁵) were transfected with the indicated plasmids for 22 h, and cells were untreated or treated with SeV (MOI = 1) for 10 h following co-immunoprecipitation and Western bolting analysis. (H) Overexpression of BAG6 impairs the endogenous interaction of VISA and TRAF2. HEK 293 cells (4×10⁶) were transfected with the indicated plasmids for 22 h, and cells were untreated or treated with SeV (MOI = 1) for 10 h following co-immunoprecipitation and Western bolting analysis. (I) BAG6-deficiency increases the endogenous interaction of VISA and TRAF2. Wild type and BAG6-deficient HeLa cells (1×10⁷) were untreated or treated with SeV (MOI = 1) for 10 h before co-immunoprecipitation and Western bolting analysis.