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

Figure 6. LGP2 interacts with mda-5 in a dsRNA-dependent manner.

Figure 6

(A) HEK293 cells were transfected with a plasmid expressing the helicase domain of mda-5 with a Flag tag (Flag.MH) and a plasmid expressing LGP2 with a V5 tag (V5.LGP2). 24 hours after transfection, cells were transfected with poly(I:C) for the indicated times. Cell extracts were subjected to immunoprecipitation with the anti-flag antibody, and proteins present in the precipitate were analysed by western blotting with anti-V5 and anti-flag antibodies. (B) Yeast cells were transformed with a plasmid expressing the helicase domain of mda-5 as a GAL4DBD fusion, and a plasmid expressing LGP2 or the indicated mutants of LGP2 as a GALAD fusion. Positive transformants were selected on SD-L-W, and growth on this media indicates that the yeast have been transformed by both plasmids. They were then streaked onto SD-L-W-H + 2 mM 3-AT and growth on this media demonstrates an interaction between the GAL4DBD fusion protein and the GAL4AD fusion protein. (C) Yeast cells were transformed with a plasmid expressing the helicase domain of mda-5 as a GAL4DBD fusion, a plasmid expressing LGP2 as a GAL4AD fusion and either the empty vector pHON7 (−), pHON7 expressing the dsRNA binding domains of PKR (PKR(1–207)) or pHON7 expressing a mutant form of PKR that is unable to bind dsRNA (M2(1–207)). Positive transformants were selected on SD-L-W-U, and growth on this media indicates that the yeast have been transformed by all three plasmids. They were then streaked onto SD-L-W-U-H + 2 mM 3-AT and growth on this media demonstrates an interaction between the GAL4DBD fusion protein and the GAL4AD fusion protein.