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).