Fig. 2. 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 (B)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 (B)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.