Figure 3.

CAD is a negative regulator of NOD2 signaling. (A) NF-κB reporter assay in NOD2-expressing HEK293T cells cotransfected with indicated amounts of CAD expression plasmid and MDP stimulated (10 ng/mL for 18 hours). Luciferase values normalized to β-galactosidase transfection control values (nLuc). Averages ±SD are shown. (B) NF-κB reporter assay in NOD2-expressing HEK293T cells cotransfected with control or CAD short hairpin RNA plasmids, MDP stimulated, and assayed as in A. Immunoblot showing RNAi-mediated knockdown of Flag-CAD expression in HEK293T cells (inset). (C) MAPK p38 reporter assay in NOD2-expressing HEK293T cells transfected with vector or CAD expression plasmids (100 ng), stimulated with MDP (100 ng/mL for 18 hours), and analyzed as in A. (D) Immunoblots of CAD expression levels in lysates from HCT116 cells grown to different densities (20 µg/lane; top panel) or immunoblots of MDP-stimulated (1 µg/mL) NF-κB p65 phosphorylation (p-p65; middle panels) or p38 phosphorylation (p-p38; bottom panels) in HCT116 cells grown to indicated density. (E) NF-κB reporter assay performed as in A in HEK293T cells expressing either NOD2 or NOD1. Cells were treated with either MDP (10 ng/mL) or the NOD1 ligand KF1B (100 ng/mL) for 18 hours. (F) NF-κB reporter assay performed as in A with cells treated with either MDP (10 ng/mL) or tumor necrosis factor α (0.1 ng/mL) for 18 hours. **P < .01, ***P < .001.