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. 2009 Dec 23;84(5):2421–2431. doi: 10.1128/JVI.02174-09

FIG. 1.

FIG. 1.

Caspase-1 and caspase-3 cleave MAVS at D429. (A) N18 cells were mock treated or adsorbed with DEN-2 (multiplicity of infection [MOI] of 5) and transfected with 2 μg of wild-type (WT) or D429A- or D429E-mutated Flag-MAVS. The cells were then incubated in the presence (+) or absence (−) of z-VAD-fmk (150 μM) for 30 h. Cell lysates were analyzed by immunoblotting, using the antibodies indicated at the left. (B and C) Recombinant Flag-MAVS-His protein (30 μg) was incubated with 2 U of active caspase-1, -3, -8, or -9 as described in Materials and Methods. The reaction mixtures were resolved by SDS-PAGE and analyzed by silver staining (B) and immunoblotting, using antibodies against Flag (lanes 1 to 5) and His (lanes 6 to 10), as indicated at the top (C). The background reactivity of these caspases with anti-His antibody was also demonstrated by immunoblotting in the absence of Flag-MAVS-His protein (lanes 11 to 14 in panel C). (D) The cleavage assay of in vitro-transcribed and -translated 35S-labeled MAVS and its mutants (D86A and D429A) was performed by coincubation with (+) or without (−) active caspase-3. The proteins were then separated by SDS-PAGE, and the signals were developed by autoradiography. (E) In vitro cleavage of Flag-MAVS-His protein by caspase-1 and caspase-3 was performed in the presence (+) or absence (−) of z-VAD-fmk (1 mM) and analyzed by immunoblotting using anti-Flag antibody. (F) N18 cells were transfected with Flag-tagged MAVS and treated with various caspase inhibitors, as indicated at the top, for 24 h. Cell lysates were harvested and analyzed by immunoblotting, using anti-Flag and anti-actin antibodies, as indicated at the left. (G) A549 cells were infected with DEN-2 (MOI of 5) and then treated with z-VAD-fmk (100 μM). Cell lysates were harvested 48 h after infection and analyzed by immunoblotting, using antibodies against endogenous MAVS, DEN-2 NS3, and actin, as indicated at the left. The open arrows indicate full-length MAVS, and the black arrows indicate cleaved MAVS. DMSO, dimethyl sulfoxide.