Behavior of the mutant Nod2 proteins. (A) Wild-type and mutant Nod2 proteins. CARDs, NOD and LRRs are indicated by black, shaded and hatched boxes, respectively. Numbers represent amino acid residues in the Nod2 protein. (B) Expression analysis of wild-type (WT) and mutant Nod2 proteins. HEK293 cells were transfected with 5 μg of plasmids producing the indicated HA-tagged Nod2 proteins. Twenty-four hours post transfection, expression of various Nod2 proteins was analyzed by immunoblot with monoclonal anti-HA antibody. (C) Response of HEK293 cells expressing mutant Nod2 proteins to CytD. HEK293 cells were transfected with 500 ng pcDNA3 and 50 ng (κB)₅LUC in the presence of 6 ng of plasmids producing the indicated HA-tagged mutant Nod2 proteins. Twenty-four hours post transfection, cells were treated (white bars) or not (black bars) with CytD (50 μM) for 7 hours before being harvested for LUC assays. Values represent the mean + s.d. of triplicate cultures. (D) Differential responsiveness of the mutant Nod2 proteins to CytD and MDP. HEK293 cells were transfected with 500 ng pcDNA3 and 50 ng (κB)₅LUC in the presence of 10 ng of plasmids producing the indicated HA-tagged mutant Nod2 proteins. Twenty-four hours post transfection, cells were untreated or treated with CytD (50 μM) or MDP (100 ng/ml, in the presence of calcium phosphate) for 7 hours before being harvested for LUC assays. Values represent the mean + s.d. of triplicate cultures.

Nod2 association with actin cytoskeleton. (A) Distribution of the Nod2 protein between Triton-X-100-soluble and -insoluble fractions. HEK293 cells were transfected with 500 ng pcDNA3 and 50 ng of plasmid expressing the HA-tagged Nod2 protein. Twenty-four hours post transfection, cells were treated or not with CytD 50 μM for 1 hour before being harvested for the preparation of Triton-X-100-soluble (S) or -insoluble (I) fractions. Each extract (S, I) was taken from equal number of cells and was separated by SDS-PAGE (10%) followed by western blotting with a rat monoclonal anti-HA or a mouse monoclonal anti-β-actin antibody. (B) Colocalization of ectopically expressed Nod2 with some specific structures of actin cytoskeleton. HEK293, Vero, Mf4/4 cells were transfected with Nod2-expressing plasmid (1 μg). Twenty-four hours post transfection, cells were stained for Nod2 with the rabbit serum (A,D,G) and for F-actin with TRITC-phalloidin (B,E,H). Images were obtained by confocal microscopy. (C,F,I) Merged F-actin and Nod2 images from panels A and B, D and E, G and H, respectively; areas of colocalization are shown in yellow. (C) Colocalization of endogenous Nod2 with some specific structures of the actin cytoskeleton. HT-29 cells were stained for Nod2 with a mixture of both rat anti-Nod2 monoclonal antibodies 7E11 (1:50) and 6F6 (1:50) and for F-actin with TRITC-phalloidin. A negative control (-) without primary antibodies was performed to test for secondary antibody cross-reaction. DIC, differential inference contrast. Diagrams depict the intensity of the fluorescence for each staining along the lines drawn on the overlay images. Arrows indicate areas of colocalization.

Both CARDs or LRRs are sufficient to target Nod2 in membrane ruffle-like structures. (A) Colocalization of the wt and mutant Nod2 proteins with F-actin in COS-7 cells. COS-7 cells were transfected with the following amounts of plasmid encoding the HA-tagged or FLAG-tagged wt or mutant Nod2 proteins: pcDNA₃-HA-wt Nod2 and pcDNA₃-HA-ΔCARDs (1 μg each), pcDNA₃-HA-ΔLRRs (4 μg), pcDNA₃-HA-CARDs (0.5 μg), pcDNA3-HA-LRRs (1 μg), pcDNA₃-FLAG-NOD (2 μg). In each case, the total DNA amount was adjusted to 4 μg with pcDNA3. Twenty-four hours post transfection, cells were stained for the wt or mutant Nod2 proteins with anti-HA mAb or anti-FLAG rabbit serum, and for F-actin with TRITC-phalloidin. Images were obtained by confocal microscopy (Leica TCS NT). Arrows indicate areas of colocalization. (B) Distribution of the mutated Nod2 proteins between Triton-X-100-soluble and -insoluble fractions. HEK293 cells were transfected with the following amounts of plasmid encoding the HA-tagged or FLAG-tagged mutant Nod2 proteins: pcDNA₃-HA-ΔCARDs (25 ng), pcDNA₃-HA-CARDs (10 ng), pcDNA3-HA-LRRs (100 ng), pcDNA₃-FLAG-NOD and pcDNA₃-HA-ΔLRRs (2500 ng each). In each case, the DNA total amount was adjusted to at least 2500 ng with pcDNA3. Twenty-four hours post transfection, cells were treated or not with CytD 50 μM for 1 hour before being harvested for the preparation of Triton-X-100-soluble (S) or -insoluble (I) fractions. Each kind of extract (S, I) from equal number of cells was separated by SDS-PAGE (10%) followed by western blotting with anti-HA or anti-FLAG mAbs.

Colocalization of wt and mutant Nod2 proteins with endogenous Rac1. (A) Rac1 colocalization with F-actin in membrane ruffles. COS-7 cells were stained for Rac1 with the anti-Rac1 mAb and for F-actin with TRITC-phalloidin. Images were obtained by confocal microscopy (Leica TCS NT). (B) COS-7 cells were transfected with the following amounts of plasmid encoding the HA-tagged or FLAG-tagged wt or mutant Nod2 proteins: pcDNA₃-HA-wt Nod2 and pcDNA₃-HA-ΔCARDs (1 μg each), pcDNA₃-HA-ΔLRRs (4 μg), pcDNA₃-HA-CARDs (0.5 μg), pcDNA3-HA-LRRs (2 μg), pcDNA₃-FLAG-NOD (2 μg). In each case, the total DNA amount was adjusted to 4 μg with pcDNA3. Twenty-four hours post transfection, cells were stained for the wt or mutant Nod2 proteins with monoclonal anti-HA or rabbit anti-FLAG antibodies and for endogenous Rac1 with a monoclonal antibody. Images were obtained by confocal microscopy (Leica TCS NT). Arrows indicate areas of colocalization.

Co-immunoprecipitation of wt and mutant Nod2 proteins with endogenous Rac1. (A) COS-7 cells were transfected with the following amounts of plasmid encoding the wt or mutant Nod2 proteins: pcDNA₃-HA-wt Nod2 and pcDNA₃-HA-ΔCARDs (1 μg each), pcDNA₃-HA-ΔLRRs (4 μg), pcDNA₃-HA-CARDs (0.5 μg), pcDNA3-HA-LRRs (2 μg). In each case, the DNA total amount was adjusted to 4 μg with pcDNA3. Twenty-four hours post transfection, cells were harvested and cell lysates were immunoprecipitated (IP) with anti-Rac1 monoclonal antibody, and proteins were visualized by western blotting with anti-HA (top and bottom panels) or anti-Rac1 monoclonal antibodies (middle panels). (B) COS-7 cells were transfected with 2 μg of pcDNA₃-FLAG-NOD. Twenty-four hours post transfection, cells were harvested and cell lysates were immunoprecipitated (IP) with anti-Rac1 or anti-FLAG monoclonal antibodies and proteins were visualized by western blotting with anti-Rac1 (lower panel) or anti-FLAG (upper panel) monoclonal antibodies. Non-specific bands corresponding to immunoglobulin heavy chain (IgH) are indicated. (C) Endogenous Nod2 and Rac1 proteins interact. HT-29 cell lysates (1 mg) were immunoprecipitated (IP) with anti-Rac1 or control mouse monoclonal antibodies and proteins were visualized by western blotting with anti-Rac1 (lower panel) or anti-Nod2 (7E11, 1:100, upper panel) monoclonal antibodies.

Nod2 localization in ribbon-like membrane ruffles induced by RacQL. (A,B) COS-7 cells were transfected with 1 μg of plasmid expressing the HA-tagged wt Nod2 protein and 2 μg of plasmid encoding the constitutively activated Rac1 mutant (Rac QL). Twenty-four hours post transfection, cells were stained for Rac1 and F-actin (A) or for Rac1 and Nod2 (B). Images were obtained by confocal microscopy (Leica TCS NT). Arrows indicate areas of colocalization.