Fig 5

Fig 5 Mylipb negatively regulates irf3 by promoting K6-linkd ubquitination of irf3 at lysine 352.

(A-B) The interaction between mylipb and irf3 protein mostly depended on the ZF domain of mylipb protein. HEK293T cells seeded in 100-mm dishes were transfected with the indicated plasmids (4 μg each). After 24 h, the cells were treated with 3-MA for 8 h. Total cell lysates were immunoprecipitated (IP) with anti-HA antibody conjugated agarose beads. Then, the immunoprecipitates and cell lysates were detected with anti-HA or anti-Flag Ab, respectively. (C) Mylipb promoted irf3 ubiquitination depended on ZF domain and ubiquitin ligase activity of mylipb. HEK 293T cells were transfected with Flag-irf3, Myc-mylipb-WT, Myc-mylipb-Δ381–416, Myc-mylipb-C381S, or empty vector, together with His-ubiquitin. At 24 h posttransfection, the cells were treated with 3-MA for 8 h. The cells were lysed using guanidinium chloride, and purifified with Ni²⁺-NTA agarose. (D) Mylipb induced the degradation of irf3 dependent of its ZF domain. HEK293T cells were transfected with Myc-mylipb-WT or its mutants, together with HA-irf3 for 24 h. The lysates were then subjected to IB with the indicated Abs. (E) Mylipb induced the degradation of irf3 dependent of its ubiquitin ligase activity. HEK293T cells were transfected with Myc-mylipb-WT or Myc-mylipb-C381S, together with HA-irf3 for 24 h. The lysates were then subjected to IB with the indicated Abs. (F) Ubiquitination assay of irf3 in mylipb-null (mylipb^−/−) zebrafish liver and their WT (mylipb^+/+) siblings. mylipb-null zebrafish (3 mpf) and their WT siblings (3 mpf) were i.p. injected with (+) or without (-) SVCV(∼2.50 × 10⁷ TCID₅₀/mL) at 10 μl/individual for 48 h, then their livers were dissected, and lysed with lysis buffer (100 μl). The supernatants were denatured at 95°C for 5 min in the presence of 1% SDS. The denatured lysates were diluted with lysis buffer to reduce the concentration of SDS (<0.1%). Denature-IP was conducted using anti-irf3 antibody and then subjected to immunoblotting with anti-Ubiquitin antibody. (G-H) Mylipb promoted K6-linked poly-ubiquitination of irf3. (I) Mylipb promoted K6-linked poly-ubiquitination at lysine 352 of irf3. (G-I) HEK 293T cells were transfected with Flag-irf3 or HA-irf3, Myc-mylipb, or empty vector, together with His-ubiquitin or its mutants. At 24 h posttransfection, the cells were treated with 3-MA for 8 h. The cells were lysed using guanidinium chloride, and purifified with Ni²⁺-NTA agarose. IB, immunoblot; KO, K-only; KR, K is mutated to R. (J) K352R mutation of irf3 abolished the inhibitory effect of mylipb on ISRE reporter activities in EPC cells. EPC cells were transfected with ISRE reporter and HA-irf3 or its mutants together with Myc-mylipb or empty vector for 24 h, and then conducted luciferase reporter activity assays. (K) K352R mutation abolished degradation of irf3 induced by mylipb. HEK293T cells were transfected with Myc-mylipb or empty vector, together with HA-irf3 or HA-irf3-K352R for 24 h. The lysates were then subjected to IB with the indicated Abs.