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. 2012 Jun 14;287(35):29213–29226. doi: 10.1074/jbc.M112.355545

FIGURE 3.

FIGURE 3.

Mapping of the domains of interaction. A, schematic representation of full-length and truncated NOD2 proteins used in co-immunoprecipitation studies. B, the deletion of both CARDs or LRRs still allows NOD2 to interact with JNKBP1. HEK293T cells were transfected with JNKBP1-V5 and HA-NOD2 WT or the truncated form (ΔCARD and ΔLRR). Lysates were immunoprecipitated with an anti-V5 antibody, and co-immunoprecipitated NOD2 proteins were revealed with an anti-HA antibody. The presence of each tagged protein was checked in whole lysates through anti-HA immunoblot (bottom). C, the CARD1 domain is sufficient to maintain the interaction of NOD2 with JNKBP1. HEK293T cells were transfected with JNKBP1-V5 and HA-CARD1 or HA-CARD2. Lysates were immunoprecipitated with an anti-V5 antibody, and Western blot analysis was performed with an anti-HA antibody. The presence of each tagged protein was checked in whole lysates through anti-HA or -V5 immunoblots (bottom). D, the NBD domain is not involved in the interaction between NOD2 and JNKBP1. HEK293T cells were transfected with JNKBP1-V5 and FLAG-NBD. Lysates were immunoprecipitated with anti-V5 antibody, and Western blot analysis was performed with an anti-FLAG antibody. The presence of each tagged protein was checked in whole lysates through anti-FLAG or -V5 immunoblots (bottom). E, the LRR domain is still able to bind JNKBP1. HEK293T cells were transfected with JNKBP1-V5 and HA-LRR. Lysates were immunoprecipitated with an anti-V5 antibody, and Western blot analysis was performed with an anti-HA antibody. The presence of each tagged protein was checked in whole lysates through anti-HA or -V5 immunoblots (bottom). F, the three main CD-associated NOD2 mutants (R702W, G908R, and L1007fs) still interact with JNKBP1. JNKBP1-V5 was cotransfected into HEK293T cells with HA-NOD2 WT, HA-R702, HA-G908, or HA-L1007fs. After 48 h, cells were harvested for protein extraction. Samples were immunoprecipitated with an anti-V5 antibody, and the NOD2 co-immunoprecipitation was analyzed by an anti-HA immunoblot. Total lysates were immunoblotted with anti-HA and -V5 antibodies. G, schematic representation of full length and truncated JNKBP1 proteins used in co-immunoprecipitation studies. H, the N-terminal WD-40 domain of JNKBP1 is essential for NOD2 binding. WT JNKBP1-V5, ΔC JNKBP1-V5, or ΔN JNKBP1-V5 was cotransfected in HEK293T cells with HA-NOD2. Lysates were immunoprecipitated with an anti-V5 antibody, and immunoblotting was performed using the indicated antibodies. The presence of each tagged protein was checked in whole lysates through anti-HA or -V5 immunoblots (bottom).