FIG. 6.
Copurification of IPS-1, RIG-I, and NS1 in detergent insoluble complexes. A total of 2 × 106 293T cells were transfected with 2.5 μg of pCAGGS-FLAG-RIG-I (lanes 2, 5, 6, and 8), 2.5 μg of pCAGGS-HA-IPS-1 (lanes 3, 5, 7, and 8), and/or 2.5 μg of pCAGGS-NS1 (lanes 4, 6, 7, and 8). Amounts of transfected DNA were adjusted with empty pCAGGS plasmid. At 48 h posttransfection, cells were subjected to reversible cross-linking reaction and then lysed in 500 μl of RIPA buffer. After centrifugation at 10,000 × g for 5 min, cell supernatants (Sup.) and pellets were separated. The supernatant was mixed with equal volume of 2 × SDS gel loading buffer. The pellet was resuspended in 50 μl of SDS gel loading buffer. The samples were boiled for 5 min and then subjected to 12% SDS-PAGE. Expression levels of Flag-RIG-I, HA-IPS-1, and NS1 were detected by Western blotting using anti-Flag, anti-HA, and anti-NS1 antibodies, respectively.