Fig. 6. 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-CARDs (1 µg each), pcDNA₃-HA-LRRs (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.