Figure 5. CypA enhances TRIM25-mediated K63-linked ubiquitination of RIG-I and facilitates recruitment of RIG-I to MAVS.
(A) Immunoblot analysis of lysates in 293T/CypA- cells transfected for 24 hr with Flag-RIG-I, along with HA-K63-Ub, HA-K48-Ub, Myc-TRIM25, or Myc-CypA, followed by immunoprecipitation with anti-Flag beads. (B) Immunoblot analysis of lysates in WT and Ppia−/− BMDMs infected with SeV for 6 hr, followed by immunoprecipitation with control mouse IgG or anti-RIG-I antibodies. Lysates and immunoprecipitation extracts were probed with K63-Ub, RIG-I and CypA antibodies. (C) Immunoblot analysis of lysates in 293T/CypA- cells transfected with Flag-RIG-I, Myc-TRIM25, or Myc-CypA for 24 hr, and immunoprecipitated with anti-Flag beads. (D) Immunoblot analysis of lysates in WT and Ppia−/− BMDMs infected with SeV for 6 hr, followed by immunoprecipitation with control mouse IgG or anti-RIG-I antibodies. Lysates and immunoprecipitation extracts were probed with RIG-I, TRIM25, CypA and antibodies. (E) Immunoblot analysis of lysates in 293T/CypA- cells transfected with Myc-CypA and Flag-RIG-I, Flag-RIG-I-C or Flag-RIG-I-N for 24 hr, and immunoprecipitated with anti-Flag beads. (F) Immunoblot analysis of lysates in WT and Ppia−/− BMDMs after SeV infection or mock-infection for 6 hr, followed by mitochondrial-cytoplasm extraction. (G) Confocal microscopy of endogenous RIG-I in 293T/CypA+ and 293T/CypA- cells stained with Mito-Tracker after SeV infection or mock infection for 6 hr. Scale bars, 10 μm. (H) Immunoblot analysis of lysates in 293T/CypA+ and 293T/CypA- cells transfected with Flag-RIG-I and Myc-MAVS for 24 hr, and immunoprecipitated with anti-Flag beads. (I) Confocal microscopy of endogenous CypA in 293T/CypA+ cells stained with Mito-Tracker after SeV infection or mock-infection for 6 hr. Scale bars, 10 μm. Data are representative of at least three independent experiments.