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. 2008 Nov 19;83(3):1465–1473. doi: 10.1128/JVI.01768-08

FIG. 4.

FIG. 4.

mda-5 and RIG-I self-associate in response to poly(I-C). 293 cells were transfected with plasmids expressing (A) myc- and Flag-tagged forms of mda-5, (B) myc- and Flag-tagged forms of RIG-I, or (C) myc-tagged RIG-I and Flag-tagged mda-5. Twenty-four hours after transfection, cells were transfected with poly(I-C) and left for a further 2 to 16 h before harvesting. Cell extracts were subjected to immunoprecipitation (IP) with an antibody against the Flag tag, and proteins present in the immunoprecipitate were analyzed by Western blotting (WB). (D) Extracts from cells transfected with myc- and Flag-tagged mda-5 (top panel) or myc- and Flag-tagged RIG-I (lower panel) were incubated with increasing concentrations of poly(I-C) in the presence of 1 mM ATP for 1 h at 4°C. Samples were then subjected to immunoprecipitation with an antibody against the Flag tag, and the presence of myc-tagged protein in the immunoprecipitate was determined by Western blotting for the myc tag. (E) Western blots were carried out on the extracts used in panel D to determine expression levels of tagged proteins.