Co-Immunoprecipitations between NOD2 and NIP candidates.

<p>NOD2 cDNA was tagged with a MYC epitope at its N-terminus and all other NIP cDNAs were tagged with EGFP. Proteins were co-expressed in HEK293T cells and 24 h after transfection, cells were lysed in a buffer containing 1% TX100. Cell lysates were first analyzed by Western blotting with anti-MYC and anti-GFP antibodies to verify the level of expression and the correct size of each protein (Total lysates). Cell lysates were then subjected to immunoprecipitation with anti-MYC (IP αMYC) and/or anti-GFP (IP αGFP) antibodies and analyzed by Western Blot with αMYC and αGFP. (A) Co-Immunoprecipitation of MycNOD2 and EGFP-RICK after αGFP immunoprecipitation and αMYC Immunoprecipitation. Negative control experiments showed that lysates containing only MycNOD2 proteins could not be immunoprecipitated with αGFP antibodies and reciprocally, lysates containing only GFP-RICK proteins could not be immunoprecipitated by αMYC antibodies (B) Immunoprecipitation experiments between MycNOD2 and 13 NIP candidates. Immunoprecipitation and Western Blot analysis are performed as in A on cell lysates coexpressing MycNOD2 and a specific GFP tagged NIP.