Figure 7.

The impact of mutation of conserved residues between the C-terminus of human NOD1 and NOD2 on receptor function. (A) Alignment of the terminal 33 amino acids of human NOD1 and NOD2. Residues highlighted in cyan are conserved across mammals in the relevant protein. The consensus sequence highlights residues found in the termini of both human NOD1 and NOD2. (B) NFκB luciferase reporter assays were performed in HEK293 cells using wild-type pCMV-NOD2 and the point mutants E1026A, E1027A, and R1037A. DNA (0.1 ng/well) was transfected into 96-well plates with (black bars) and without (white bars) muramyl dipeptide (MDP). After 24 h cells were lysed and NFκB activity determined. Results show the average of three independent experiments and *p < 0.05. Error bars indicate SEM. (C) Subcellular fractionation was performed with wild-type and mutant NOD2 constructs to separate the cytoplasmic (C) and membrane-bound (M) fractions. Proteins were identified with the specified antibodies. Blots are representative of three independent experiments.