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. 2008 Jul 4;283(27):18621–18626. doi: 10.1074/jbc.M801451200

FIGURE 3.

FIGURE 3.

CYLD physically interacts with RIG-I and inhibits the ubiquitination and signaling function of RIG-I. A, ubiquitination of transfected RIG-I. 293 cells were transfected with (+) or without (–) FLAG-tagged RIG-I either in the absence (–) or presence of wild type (Wt) CYLD or a catalytically inactive CYLD mutant (Mut, CYLD residues 1–932). All cells were also transfected with HA-tagged ubiquitin (Ub). RIG-I was isolated by IP using anti-FLAG, and the ubiquitin-conjugated RIG-I was detected by IB using anti-HA (upper panel). The level of RIG-I expression was monitored by IB (lower panel). B, ubiquitination of RIG-I mutant. 293 cells were transfected as in A except for the replacement of wild type RIG-I with a RIG-I truncation mutant encoding its N-terminal 2 CARD domains (2CARD). The ubiquitination and expression of RIG-I(2CARD) were analyzed as in A. C, ubiquitination of endogenous RIG-I. Endogenous RIG-I and MAVS were isolated by IP from lysates of CYLD+/+ and CYLD–/– DCs, and the ubiquitinated RIG-I and MAVS were detected by IB using anti-ubiquitin (upper panel). As a control, parallel ubiquitination of RelA was analyzed using MG132-treated DCs. Cell lysates were subjected to IB to monitor the expression of RIG-I, MAVS, and RelA. D, CYLD+/+ and CYLD–/– MEFs were transfected with (+) or without (–) the indicated expression vectors along with IFNβ-luc and the control reporter pRL-tk-luc. After 36 h, cell lysates were subjected to dual luciferase assays. The IFNβ-specific luciferase activity was normalized on the basis of the control Renilla luciferase and presented as in Fig. 1D. E, 293 cells were transfected with HA-CYLD along with an empty pcDNA vector, FLAG-RIG-I, FLAG-2CARD, FLAG-MAVS, or HA-TRAF3. The expressed proteins were isolated by IP using anti-FLAG (for RIG-I, 2CARD, and MAVS) or anti-TRAF3 (for TRAF3), and the co-precipitated CYLD was analyzed by IB using anti-HA (top panel). The membranes were reprobed with anti-FLAG or anti-HA to detect the expression of RIG-I and 2CARD (left, middle panel), MAVS (right, 2nd panel), and TRAF3 (right, 3rd panel). The cell lysates were subjected to direct IB to analyze CYLD expression (bottom panel).