Figure 8.

IRF-3 interaction activates Bax and causes Cyt c release from the mitochondria. (A) HT1080 (Wt) and HT1080-siIRF-3 (siIRF-3) cells were transfected with poly(I:C) for 6 h, after which the cell lysates were immunoprecipitated with Bax 6A-7 antibody and the immunoprecipitates were analysed by western blot using polyclonal Bax antibody. (B) Purified human IRF-3 was used for Bax oligomerization assay as described in Materials and methods; at the end of the reaction, the mitochondrial pellet was extracted in HE buffer containing 1% CHAPS and the extract was applied to Superdex 200 PG XK16/60 column; the fractions were analysed by western blot for Bax; column calibration was performed using the molecular weight standards kit as indicated. (C) Caspase-8-cleaved Bid (Bid, R&D Systems, 50 nM) was used for mitochondrial activation assay using the mitochondria-free cytosolic extract from Wt or Bax KO MEF cells as described in Materials and methods; at the end of the reaction, the mitochondrial pellet and the supernatant (sup) were analysed for released Cyt c, as indicated. (D) Purified activated human IRF-3 (500 nM) was used in mitochondrial activation assay using the mitochondria-free cytosolic extract from Wt or Bax KO MEF cells and analysed as described in (C). (E) Bid (50 nM) was used for in vitro Cyt c release assay using recombinant human Bax (120 nM) as described in Materials and methods; at the end of the reaction, the supernatant (sup) and the pellet were analysed for Cyt c by western blot. (F) Purified activated IRF-3 (500 nM) was used for in vitro Cyt c release assay using recombinant human Bax (120 nM) and analysed as described in (E).