Cloning of constructs.

<p>GenBank <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004931#pone.0004931-Benson1" target="_blank">[66]</a> accession numbers, featured domains as well as corresponding amino acid residues are indicated, respectively.</p>*<p>NOD2 tripled mutation – E69K, D70K, D71K; CARD: caspase activation and recruitment domain, PYD: pyrin domain.</p

NOD2 interaction with distinct NLRP members.

<p><i>Upper panel</i>, A “+” indicates an interaction, “−” symbolizes no interaction (n.d.: not done). A short isoform of NOD2 (NOD2-S, residues 1–180), maintains the interaction with NLRP1, -3, and -12. Furthermore, a linker region within NLRP1 (residues 92–341) proved sufficient for interaction with NOD2. <i>Lower panel</i>, Schematic illustration of particular NOD2/NLRP1 constructs and their respective interactions. FIIND: Function to find domain.</p

The interaction matrix.

<p>In an “each against all” approach an overall number of 676 (26×26) effector domain combinations were analyzed. A “+” indicates an interaction between a particular pair, whereas “−” symbolizes no interaction. In total, the approach yielded 25 distinct associations, which actually corresponded to 7 unique pairs of interacting proteins, as well as 5 homodimerizations.</p

NLR protein-protein interactions.

<p>Respective protein-protein interactions were mined from the scientific literature or retrieved from MiMI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004931#pone.0004931-Jayapandian1" target="_blank">[65]</a>. Results represent data from our yeast two-hybrid analysis (interaction observed or not observed). Baits and preys indicate individual domains tested in our analysis with interacting constructs in bold. References to reported interactions are specified.</p>§<p>Initially not observed by “each against all” approach, but observed subsequently with refined constructs.</p>*<p>No data on a direct interaction.</p

Interaction of distinct NOD2 mutants with RIPK2.

<p><i>Left panel</i>, Yeast two-hybrid analysis. NOD2 CARD1+2 interacts with RIPK2 CARD (column 1). No interactions were observed for NOD2 variants harboring a disrupted acidic patch (transformation 2, 3, and 4). An unrelated mutation not affecting the interaction interface had no effect (column 5). <i>Right panel</i>, Autoradiography of an <i>in vitro</i> transcription/translation of NOD2 proteins. NOD2 constructs were expressed using similar amounts of DNA and lysates.</p

NOD2 directly interacts with NLRP1, NLRP3, and NLRP12.

<p><i>Left panel</i>, Yeast two-hybrid analysis of NOD2 CARDs (residues 1–267) showed interaction with NLRP1,-3 and -12, but not with other NLRP proteins (NLRP2, -7, -10, or -11; column 1). Lam c was used as a negative control (column 2). Interestingly, NOD2 mutant E69K maintained the binding to NLRP1, -3, and -12 (column 3), whereas NOD2 D70K (column 4) as well as the NOD2 triple mutant (3xmut, column 5) did not. An unrelated mutation not located within the prospective interaction interface had no effect (column 6). <i>Right panel</i>, Physical interaction of NOD2 and NLRP3 in human cells. Western analysis of lysates (IN) and immunoprecipitated complexes (IP) from HEK293T cells, transiently transfected with expression plasmid encoding human HA-NOD2 and FLAG-NLRP3. NLRP3 was immunoprecipitated from cell lysates using a FLAG-epitope specific antibody. Proteins were detected using anti-HA and anti-FLAG antibodies, respectively. As negative control, proteins were immunoprecipitated with FLAG-epitope specific antibody from lysates of HEK293T cells transiently transfected with HA-NOD2, but not FLAG-NLRP3.</p

Strength of selected interactions in terms of HIS3 reporter gene activation.

<p>The maximum concentration of 3-aminotrizol supporting visible growth of transformants is indicated.</p

RIPK2 CARD forms homodimers/-oligomers.

<p><i>Left panel</i>, Yeast two-hybrid analysis revealed that RIPK2 CARD (residues 427–527) forms homodimers/-oligomers (column 1 on SD/-4). Lamin c (Lam c) was used as control (column 5). <i>Right panel</i>, GST pull down assay. Specific binding of ³⁵S-labeled RIPK2 CARD (residues 427–527) was observed to recombinant expressed GST-RIPK2 CARD (residues 427–527), whereas binding to GST-Lam c was not detected. SD/-2: SD/-Leu/-Trp, SD/-4: SD/-Ade/-His/-Leu/-Trp.</p

Role of BAT3 for iDCs and the effect of purified BAT3 on NK cell function.

<p>(A) Standard Europium release assay: Inhibition of NK-dependent lysis of iDCs in the presence of anti-BAT3 was significant (paired t-test, p-value = 0.008). (B) iDCs transfected with either control siRNA or BAT3 siRNA were co-incubated with NK cells for 4 hours at 37°C. The decrease of iDC lysis upon BAT3 down regulation was significant (p = 0.01). (C) Lysis of mature DCs upon pre-incubation with control antibodies, anti-HLA-ABC and/or anti-BAT3. (D) Co-culture of iDCs with activated NK cells at 5∶1 ratio (iDC∶NK) promotes the maturation of iDCs as shown by FACS analysis to detect expression of the maturation marker CD86. Inhibition of this effect is achieved by soluble purified BAT3. The y-axis represents the mean fluorescence intensity (MFI). (E) The lysis of Raji cells is inhibited by soluble BAT3 and anti-NKp30 compared to the control protein His BB4. The decrease of the lysis was significant (paired t-test, p value = 0.019). (F) NK cells were pre-stimulated with immobilized HisBB4 (control) and purified BAT3 prior a cytotoxicity assay with Raji cells as targets at different effector : target cell ratios. NK cells were derived from different donors for each experiment. Error bars for the lysis experiments represent standard deviation of three replicates. One representative experiment of four is shown.</p

Bio-chemical characterization of the released BAT3.

<p>(A) Detection of BAT3 expression on exosomes by electron microscopy {left panel: gold antibody control (140000×) and right panel: exosomes stained with anti-BAT3 antibody (140000×)}. (B) Western blotting to detect BAT3, Hsp70, Lamp-2 and CD9 in exosomal fractions (30 µg) and lysate (10 µg) of 293T cells and iDCs. (C,D) FACS analysis to detect BAT3 and various surface markers on exosomes, that were purified from iDCs (C) or 293T cells (D) that were immobilized to latex beads. Grey background represents isotype control. (E) FACS analysis of exosomes derived from control transfected (wt) or BAT3-transfected (BAT3) 293T cells revealed over-expression of BAT3 on the exosomal surface. Specific binding of anti-BAT3, NKp30-Ig and NKp46-Ig was detectable. Grey histograms: background (secondary antibody) staining of beads coated with exosomes. (F) Western blot analysis demonstrates that the enhanced secretion of BAT3 into the supernatant obtained from tumor cells (293T) when treated with heat shock (HS, lane: 3) or left untreated (UT, lane: 2). Lanes 4 and 5 demonstrate the co-immunoprecipitation of BAT3 by using either a polyclonal BAT3 antibody (4^th lane) or a monoclonal Ab against Hsp70 (5^th lane). The western blot is stained for BAT3. Lane 1 (M) indicates the marker.</p