Fig. 6

Zebrafish and human LGP2s play antithetic roles in regulating MDA5-/RIG-I-triggered IFN response in fish cells dependently of the exact doses of MDA5/RIG-I (A–C) Zebrafish LGP2 bound to DrMDA5 (A), DrRIG-I (B) or DrMAVS (C) independently of poly(I:C) by Co-IP assays. HEK293T cells seeded in 10 cm dishes overnight were transfected with LGP2-myc, together with Flag-DrMDA5 (A), Flag-DrRIG-I (B), DrMAVS-Flag (C) (5 μg each), in the presence or absence of poly(I:C) at 200 ng/ml) (A–C). Cell lysates were immunoprecipitated with anti-Flag Ab, followed by western blot analysis of the immunoprecipitates with anti-myc Ab. (D) In vitro-translated DrLGP2 bound to in vitro-translated DrMDA5 and DrRIG-I by pull-down assays. 50 μl of in vitro-translated DrLGP2-Flag was incubated with in vitro-translated DrMDA5-HA or in vitro-translated DrRIG-I-HA in NP40 lysis buffer at 4°C for 12 h. The bead-bound protein complex was performed by western blotting analysis with anti-Tag Ab. In vitro-translated DrMAVS-Flag was incubated with in vitro-translated DrRIG-I-HA as positive control, and with GFP-HA as negative control. (E–G) DrLGP2 and HsLGP2 downregulated IFN promoter activation in fish cells induced by DrMAD5 or DrRIG-I at a high dose. EPC cells seeded in 24-wells plates were transfected with DrIFNφ1pro-luc or CaIFNpro-luc (E, F) or HsIFNβpro-luc (G), DrMAD5 or DrRIG-I (200 ng each), DrLGP2 or HsLGP2 at increasing doses (0, 10, 50, 100, 200 ng) for 24 h, followed by luciferase assays. P values were calculated using ANOVA. **P<0.01, *P<0.05. (H, I) Titration of DrMDA5 and DrRIG-I revealed DrLGP2-mediated antithetic regulation of IFN response in fish cells. CO cells seeded in 24-well plates were transfected with CaIFNpro-luc (200 ng), DrLGP2 at 5 ng (H) or at 200 ng (I), DrMDA5 or DrRIG-I at increasing doses for 24 h, and finally collected for luciferase assays.