Figure 7.

Laboratory of genetics and physiology 2 (LGP2) inhibits the K63-linked ubiquitination of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). (A,B) Grass carp reovirus (GCRV) infection upregulates the K63-linked ubiquitination of RIG-I and MDA5. Fathead minnow (FHM) cells were transfected with 2 µg HA-Ub-K63O, 6 µg RIG-I-Flag (A), or 6 µg MDA5-Flag (B) in 10 cm² dishes, respectively. At 24 h post-transfection, the cells were treated with MG132 and GCRV for 6 and 12 h. Then, the cells were harvested for immunoprecipitation (IP) with anti-Flag antibody (Ab) and IBs with anti-HA and anti-Flag Ab. (C,D) LGP2 inhibits the K63-linked ubiquitination of RIG-I and MDA5 in a dose-dependent manner. FHM cells were seeded in 10 cm² dishes for 24 h and transfected with 1 µg HA-Ub-K63O, LGP2 (0, 1.5, and 3 µg) together with decreasing amounts of empty vector (3, 1.5, and 0 µg), 4 µg RIG-I-Flag (C), or 4 µg MDA5-Flag (D). At 24 h post-transfection, the cells were treated with MG132 and GCRV for 6 h. Then, the cells were harvested for IP with anti-Flag Ab and IBs with anti-HA and anti-Flag Ab, respectively. (E,F) LGP2 represses the K63-linked ubiquitination of RIG-I CARDs, but not MDA5 CARDs. FHM cells were transfected with 1 µg HA-Ub-K63O, LGP2 (0, 1.5, and 3 µg), empty vector (3, 1.5, and 0 µg), 4 µg RIG-I-CARD-Flag (E), or 4 µg MDA5-CARD-Flag (F) in 10 cm² dishes. At 24 h post-transfection, the cells were treated with MG132 and GCRV for 6 h. Then, the cells were harvested for IP with anti-Flag Ab and IBs with the indicated Abs.