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. 2013 May 9;8(5):e64202. doi: 10.1371/journal.pone.0064202

Figure 2. LGP2 stimulation of poly(I:C) signaling is dependent upon endogenous mda-5.

Figure 2

HEK293 cells were transfected with the IFN-β reporter plasmid, the β-galactosidase expression plasmid, either the empty vector pEFpl2 or pEF.LGP2, and either a control siRNA (A), an siRNA directed against RIG-I (B), or an siRNA directed against mda-5 (C). 24 hours after transfection cells were further transfected with the indicated amounts of poly(I:C) for 16 hours. Cell lysates were analysed for luciferase and β-galactosidase activity, and relative expression levels calculated. (D) The effectiveness of siRNAs against RIG-I and mda-5 was tested by immunoblotting. HEK293 cells were transfected with a vector expressing Flag-tagged RIG-I and either a control siRNA or the RIG-I siRNA (upper panel). Cells transfected with either the control siRNA or the mda-5 siRNA were exposed to IFN for 16hrs to induce mda-5 expression (lower panel). Cell extracts were subjected to immunoblotting with either anti-Flag (for RIG-I detection), anti-mda-5 or anti-tubulin as a loading control.