Fig. 4

Figure 4. Zebrafish TRIM25 Activate RIG-I through K63-Linked Ubiquitination on Its 2CARD Domain (A) Interaction between zebrafish TRIM25 and RIG-I 2CARD domain. Whole-cell lysate (WCL) of embryos microinjected with TRIM25 and FLAG-RIG-I (left panel), mutant FLAG-RIG-I(Δ2CARD) (middle panel), or FLAG-2CARD domain (right panel), upon stimulation with poly(I:C) or mock PBS, were used for IP and IB as indicated. (B) Function of TRIM25 as a E3 ubiquitin ligase of RIG-I. WCL of embryos microinjected with Ub-HA, TRIM25-Myc, and FLAG-RIG-I or FLAG-RIG-I(Δ2CARD), upon stimulation with poly(I:C) or mock PBS, were used for IP and IB. (C) 2CARD domain could be ubiquitinated by TRIM25. WCL of embryos microinjected with Ub-HA, TRIM25-Myc, and FLAG-2CARD, upon stimulation with poly(I:C) or mock PBS, were used for IP and IB. (D) Knockdown of endogenous TRIM25 in vivo perturbs 2CARD ubiquitination. WCL of embryos microinjected with Ub-HA and FLAG-2CARD, as well as TRIM25MO or CtlMO, were used for IP and IB. (E) Pattern of K63-linked ubiquitination of RIG-I mediated by TRIM25. After co-microinjection with FLAG-RIG-I and TRIM25-Myc, together with wild-type (WT) Ub-HA or mutant Ub-HA with K48R or K63R, embryo WCLs were used for IP and IB. WCLs were used for IB with anti-Myc to show TRIM25 expression and anti-β-actin for loading control. The relative grayscale value of anti-HA was normalized to that of anti-FLAG within each treatment group. The data in the control groups were arbitrarily set to 1.