FIG. 4.

Caspase-cleaved MAVS loses the ability to trigger cell death. (A) (Top) The expression pattern of each MAVS construct in N18 cells was analyzed using an immunofluorescence assay 16 h after transfection. Green, staining with anti-Flag antibody plus Alexa Fluor 488-labeled secondary antibody; blue, nuclear 4′,6-diamidino-2-phenylindole [DAPI] staining. (Bottom) Cell morphology was also photographed by phase-contrast microscopy 30 h after transfection. (B) Twenty-four hours after transfection, the transfected cells were collected and analyzed for annexin V positivity by flow cytometry. (C) The culture supernatants were collected 30 h after transfection, and cytotoxicity was estimated by measuring LDH release. The results are expressed as averages and standard deviations (n = 2 per group). (D) MAVS protein expression and caspase-3 activation were determined in N18 transfectants by immunoblotting with antibodies against the Flag tag and caspase-3, as indicated at the left. Flag-tagged GFP was used as a protein expression control. (E) The levels of caspase-3/7 activation triggered by various MAVS constructs were determined in 293FT cells transfected with the GFP control or the indicated MAVS constructs for 48 h, using Caspase-Glo 3/7 assays. The change in induction relative to that of the GFP control was determined, and the results are expressed as averages and standard deviations (n = 3 per group).