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. 2010 Apr 20;285(26):20252–20261. doi: 10.1074/jbc.M109.089912

FIGURE 4.

FIGURE 4.

RIG-I CARD interaction with TRIM25 and MAVS CARD-PRD. A, S8D and S8E RIG-I 2CARD mutants exhibit a decreased TRIM25 binding ability. Top, 293T cells (2 million cells) were co-transfected with the indicated plasmids. 24 h after transfection, the interactions between TRIM25 and RIG-I 2CARD WT or mutants were analyzed by immunoprecipitation (IP), followed by Western blot (WB). Bottom, Western blot showing the expression levels of the transfected proteins and tubulin. B, RIG-I phosphorylation at serine 8 decreases its ability to bind TRIM25. At 48 h after transfection with vector or HA-TRIM25 together with RIG-I-FLAG, HEK293T cells were treated with 100 nm calyculin A (Invitrogen) for 45 min. WCLs were subjected to immunoprecipitation with α-HA. Immunoprecipitates and WCLs were then immunoblotted with anti-Ser(P)8 (pSer8)-RIG-I, anti-FLAG, or anti-HA antibodies. C, S8D and S8E RIG-I 2CARD mutants have a decreased ability for MAVS binding. Top, 293T cells (2 million cells) were co-transfected with the indicated plasmids. 24 h after transfection, CARD interaction with the MAVS CARD-PRD region was analyzed by immunoprecipitation followed by Western blot. Bottom, Western blot showing the expression levels of the different transfected proteins and tubulin.