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.</p

Fold regulation by MDP or LPS in THP-1 cell line.

<p>Fold regulation by MDP or LPS in THP-1 cell line.</p

Interaction between NIPs and NOD2 mutants.

<p>(A) Interaction between 8 full length NIPs and different NOD2 mutants (Blau or CD associated mutations) were tested and scored by Y2H using three reporter genes (lacZ/HIS3 and URA). (B) BRET titration experiments (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165420#pone.0165420.g003" target="_blank">Fig 3</a>) compared the interaction of NOD2WT and NOD2FS mutant (1007fs) with RICK, CHMP5 and TRIM41.</p

<i>VIM</i> results for haplotypes with frequency >0.05.

<p><i>VIM</i> results for haplotypes with frequency >0.05.</p

A NOD2 interactome network including new NIPs and possible functions: This partial network includes the new NIPs isolated in this study and some other known NOD2 partners issued from public protein/protein interaction databases (BIOGRID3.4, STRING) [69, 77].

<p>For clarity, not all proteins involved in the NOD2 network were represented but only the new NIPs identified in this study and some NOD2 partners interacting with these new NIPs. Red filled circles represent new NIPs isolated here. Blue filled circles represent already known NIPs. Bold circles represent NIPs also characterized in other published studies.</p

TDT results for DOCK7, GOLGB1 and IKBIP for haplotypes with frequency >0.05.

<p>TDT results for DOCK7, GOLGB1 and IKBIP for haplotypes with frequency >0.05.</p

Summary of Y2H, co-IP and BRET studies.

<p>Summary of Y2H, co-IP and BRET studies.</p

Interaction between NOD2 and NIP by BRET.

<p>BRET titration experiments were performed by fixing the amount of the donor protein (fused to renilla Luciferase) and by increasing the amount of the acceptor protein (fused to EYFP) coexpressed by transient transfection in HEK293T cells. A specific energy transfer can be detected when the two proteins interact directly (i.e. within a distance of 10 nm or less) and must increase hyperbolically as a function of the acceptor/donor (Fluorescence/Luminescence) ratio. In comparison, non-specific interactions and random collisions would increase linearly. Regression curves assuming one binding site are represented as BRET value (mBRET unit) as a function of the Fluorescence/Luminescence ratio. In these BRET titration experiments there are no unit for the x-axis since it is a ratio of protein measuring the relative expression of each BRET protein partner (ratio between the acceptor -fused to EYFP and the donor-fused to luciferase). BRET saturation (BRETmax value) is reached when all expressed donor proteins are involved in an interaction with an acceptor protein. BRET experiments only provide a relative affinity for proteins (apparent Kd). The relative affinity between 2 proteins (apparent Kd) corresponds to the BRETmax/2 value (BRET50). Experiments were performed at least 3 times and a representative experiment is shown. All titration regression curves (in absence of MDP) fitted with a R² coefficient >0.98 except for LucNOD2 / EYFPDOCK7 (R² = 0.9621). (A) BRET titration experiments between donor LucRICK and acceptor EYFPNOD2 in absence (control) or in presence of a 22h MDP stimulation (10μg/ml). Negative control BRET experiments were performed with the LucRICK donor replaced by unfusioned Luciferase (middle panel) or with the acceptor EYFPNOD2 replaced by EYFP (right panel). (B) BRET titration experiments between NOD2 and 9 NIP candidates in absence (control) or in presence of MDP for a 18-22h stimulation. (C) BRET titration experiments between NIPs candidates and NOD2 showing no specific BRET saturation signal: Non-specific BRET linear profile for NOD2/VIM (left), and BRET values below threshold (50 mBRET Unit) for NOD2/IKBIP (middle) and NOD2/KRT15 (right).</p

Supplementary_Material – Supplemental material for Technical Validation of a Reverse-Transcription Quantitative Polymerase Chain Reaction In Vitro Diagnostic Test for the Determination of MiR-31-3p Expression Levels in Formalin-Fixed Paraffin-Embedded Metastatic Colorectal Cancer Tumor Specimens

<p>Supplemental material, Supplementary_Material for Technical Validation of a Reverse-Transcription Quantitative Polymerase Chain Reaction In Vitro Diagnostic Test for the Determination of MiR-31-3p Expression Levels in Formalin-Fixed Paraffin-Embedded Metastatic Colorectal Cancer Tumor Specimens by Lucas Ramon, Catherine David, Karine Fontaine, Elodie Lallet, Charles Marcaillou, Séverine Martin-Lannerée, Virginie Decaulne, Céline Vazart, Anne-Héloise Gélibert, Raouf Ben Abdelali, Jean-Marc Costa, Francis Rousseau, Raphaële Thiébaut, Larry Yost and Yann Gaston-Mathé in Biomarker Insights</p