Expression and cellular localization of endogenous NOD2. (A) Western blot analysis using rabbit NOD2 antiserum HM2559 and anti–β-actin. 10 μg of total protein from different intestinal epithelial cells (IEC), COS7, and HEK293 cell lines were loaded onto 4–12% Tris-glycine gel. (B) NF-κB activation in HT29 and Caco-2 cells after stimulation with 1 μg/ml MDP-LD or MDP-LL using NF-κB luciferase reporter assay and normalization with renilla after 18 h of transfection. Error bars represent SEM of at least four separate experiments. *, P < 0.05. (C) Confocal microscopy examination of endogenous NOD2 in HT29 and Caco-2 cells using rabbit NOD2 antiserum HM2559 or preimmune serum and anti–rabbit Texas red as a secondary antibody. Cytosolic and surface membrane localization (arrows) of NOD2 in HT29 cells is shown. Bar, 20 μm.

Membrane association of expressed NOD2. (A) Amino acid sequences of the COOH-terminal domain of NOD2 and the NOD2 3020insC mutant, which is associated with CD. (B) Confocal microscopy examination of Caco-2 cells that are transfected with Flag-NOD2, Flag-NOD2 3020insC mutant, and empty vector (pCMVtag2C) shows the membrane association of NOD2 (arrows) but not of the NOD2 mutant. NOD2 and 3020insC mutant were detected by using monoclonal anti-Flag antibody followed by fluorescein-conjugated anti–mouse IgG. (C) Western blot analysis using anti-Flag, anti–E-cadherin, or anti–lactate dehydrogenase antibodies. Caco-2 cells were transfected with Flag-NOD2 (WT) or Flag-NOD2 3020insC (mutant). Cytosolic (C) and membrane (M) fractions were separated as described in Materials and methods. Proteins were fractionated through 4–12% Tris-glycine SDS-PAGE and were subjected to Western blot analysis by using anti-Flag antibody to detect NOD2 expression. The ratio in the membrane and cytosolic fractions that were determined after the quantification of NOD2 3020insC mutant was compared with NOD2 wild type. The result is the mean of four separate experiments. Error bar represents SEM. *, P < 0.05. (D) Caco-2 cells were cotransfected with GFP-NOD2 and Flag-NOD2 or with GFP-NOD2 and Flag-NOD2 3020insC mutant and were detected using anti-Flag antibody for Flag-tagged constructs. Only the NOD2 wild type showed plasma membrane association (arrows). (E) Caco-2 cells that were transfected with GFP-NOD2 were stained with anti–E-cadherin antibody, a membrane marker, to confirm the plasma membrane association of the NOD2 wild type. Bars, 20 μm.

NOD2 membrane association is COOH-terminal dependent. (A) Sequences of Flag-NOD2 COOH-terminal deletion and substitution mutants. (B) Caco-2 cells were transfected with Flag-NOD2 mutant constructs. Mutants 1–12 were detected by confocal microscopy using monoclonal anti-Flag antibody followed by fluorescein-conjugated anti–mouse IgG. Membrane association is still observed for some NOD2 mutants (arrows). (C) COS7 cells were transfected with GFP-NOD2 3020insC and were stained with calnexin (ER marker). Some of the observed vesicles colocalized with calnexin (arrows). Bars, 20 μm.

Ligand-induced NF-κB activation and IL-8 release are dependent on NOD2 membrane association. (A) Expression of NOD2 and mutants without tag that were transfected into HEK293 cells was determined by Western blot analysis using NOD2 antiserum HM2563. (B) HEK293 cells were transfected with 1 ng NOD2 expression vector or mutants together with 1 μg MDP-LD. NF-κB activity was determined by using an NF-κB luciferase reporter assay and was normalized with renilla after 18 h of transfection. Fold increase of NF-κB activation was determined by comparing untransfected and nonstimulated HEK293 with MDP. (C) IL-8 released in the supernatant of HEK293 cells that were transfected with mutants (M) of NOD2 and stimulated 18 h with 1 μg MDP-LD was measured by ELISA. Error bars represent SEM of at least four separate experiments. NT, nontransfected. *, P < 0.05.

Ligand-induced NF-κB activation are dependent on a three–amino acid motif that is required for membrane association. (A, 1) Amino acid sequences of Flag-NOD2 COOH-terminal substitution mutants. (2) Expression of these mutants was determined by Western blot analysis using NOD2 antiserum HM2563. (3) Fold increase of NF-κB activation was determined as described in Fig. 4. (B, 1) GFP-NOD2 wild type and the three main NOD2 mutations that are associated with CD (GFP-NOD2 3020insC, GFP-NOD2 R702W, and GFP-NOD2 G908R) were transfected in COS7 cells. Only NOD2 3020insC failed to colocalize with the plasma membrane, whereas the two other NOD2 mutant forms still showed membrane association (arrows). Bar, 20 μm. (2) Expression of NOD2 mutants and NF-κB activation were determined and compared with untransfected and nonstimulated HEK293 with MDP-LD, as described in Fig. 4. Error bars represent SEM of at least four separate experiments. *, P < 0.05.