Figure 5C - Peptides eluted from MHC class I molecules in Hela+IFNy and Hela-TAPBPR KO+IFNy

Peptide-MHC class I complexes were isolated by affinity chromatography using W6/32 from IFN-γ treated HeLa and HeLa-TAPBPR KO cells. Eluted peptides were analysed using LC-MS/MS. Peptides were assigned as HLA-A*68:02 and HLA-B*15:03 binders based on their peptide motifs using the online programme NetMHC

Figure 5 - Figure supplement 1 - Peptides eluted from HeLa-S+IFNy and HeLa-S-shTAPBPR+IFNy

Peptide-MHC class I complexes were isolated by affinity chromatography using W6/32 from IFN-γ treated HeLa-S cells and IFN-γ treated HeLa-S cells depleted of TAPBPR using shRNA. Eluted peptides were analysed using LC-MS/MS. Peptides were assigned as HLA-A*68:02 and HLA-B*15:03 binders based on their peptide motifs using the online programme NetMHC

Figure 5D - peptides eluted from HeLa and HeLa-WT-TAPBPR cells

Peptide-MHC class I complexes were isolated by affinity chromatography using W6/32 from HeLa and HeLa overexpressing WT-TAPBPR. Eluted peptides were analysed using LC-MS/MS. Peptides were assigned as HLA-A*68:02 and HLA-B*15:03 binders based on their peptide motifs using the online programme NetMHC

Figure 6E - Peptides eluted from HLA-B & -C from WT, TAPBPR depleted and tapasin deficient KBM-7 cells

HLA-B and -C complexes were isolated using affinity chromatography using B1.23.2 from IFN-γ treated WT, TAPBPR stably depleted (shTAPBPR) and tapasin deficient (TPN-) KBM-7 cells. Eluted peptide were analysed using LC-MS/MS

Figure 6B - Peptide eluted from HLA-A2 from WT, TAPBPR depleted, and tapasin deficient KBM-7 cells

Peptide loaded HLA-A2 complexes were isolated using affinity chromatography with BB7.2 from IFN-γ treated WT, TAPBPR stably depleted (shTAPBPR) and tapasin deficient (TPN-) KBM-7 cells. Eluted peptide were analysed using LC-MS/MS

NOD2 is ubiquitinated.

<p>A) To determine NOD1 and NOD2 ubiquitination, denatured lysates of HEK293T cells expressing the indicated proteins were subjected to immunoprecipitation. Immunoprecipitates were immunoblotted using the indicated antibodies B) To determine the type of ubiquitin-linkage, lysates of HEK293T cells expressing the NOD1 or NOD2 were subjected to immunoprecipitation. Endogenous ubiquitin was revealed using a K48-link specific antibody. C) HEK293T cells were transfected for 72 h with siCTR, siTRIM27-1 or -3 after expression of Flag-NOD2 and HA-Ubiquitin. Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. D) TRIM27 and a E3 mutant of TRIM27 were expressed together with NOD2 and HA-ubiquitin in HEK293T cells. Immunoprecipitates were probed with the indicated antibodies. E) HEK293T cells expressing the indicated proteins were stimulated with 10 µM MDP for the indicated time. Lysates were subjected to immunoprecipitation as described in (C). Densitometric analysis of the TRIM27 signal in the immunoprecipitation normalized to the input signal is shown (bottom). Representative data of at least three independent experiments are shown.</p

TRIM27 contributes to proteasomal degradation of NOD2.

<p>A) HEK293T cells transfected with low amounts of Flag-NOD2 and myc-TRIM27, E3 or CTR as indicated were treated with 30 µg/ml cycloheximid (CHX) and immunoblots of total cell lysates (top) were performed using the indicated antibodies. GAPDH served as loading control. B) HEK293T cells expressing the indicated proteins were treated with 100 nM bortezomib. Lysates were subjected to immunoprecipitation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041255#pone-0041255-g001" target="_blank">Figure 1A</a>. Actin served as loading control. C) HEK293T cells transfected for 48 h with siCTR, siTRIM27-1 or -3 and subsequently with Flag-NOD2 were treated with 30 µg/ml CHX, as indicated. Immunoblots of total cell lysates were performed using the indicated antibodies. GAPDH served as loading control. Representative data of at least three independent experiments are shown (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041255#pone.0041255.s003" target="_blank">Fig. S3B</a>).</p

TRIM27 negatively regulates NOD2 signaling.

<p>A–C) NF-κB luciferase assays in HEK293T cells to determine the influence of TRIM27 overexpression on MDP-induced NOD2-mediated (A), TNF- (B), or IKK-β-induced (C) NF-κB activation. Normalized luciferase activity (nRLU) of unstimulated (white bars) and stimulated (black bars) samples is shown. Values are given as mean+SD (n = 3). *, P<0.05; ***, P<0.005. <i>D.</i> Sections from human colon derived from healthy and active Crohn's disease patients. Staining with DAPI (blue), phalloidin-FITC (green) and with α-TRIM27 antibody (red) is shown. Staining with a rat serum is shown as control in the lower panel (rat serum). Slides are representative for 3 control and 4 Crohn's disease patients. Scale bar = 20 µm. <i>E.</i> qRT-PCR of TRIM27 mRNA expression in colonic biopsies derived from patients with Crohn's disease and controls (CTR) with no evidence of mucosal inflammation. Each symbol represents one patient. TRIM27 mRNA expression normalized to GAPDH is shown. *, P<0.05 (n = 6).</p

NOD2 physically interacts with TRIM27 via the NBD domain.

<p>A–C) Lysates of HEK293T cells expressing the indicated proteins were subjected to immunoprecipitation using anti-Flag (A and C) or anti-myc beads (B). Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. D) Immunoprecipitation of endogenous NOD2 from SW480 cells using the NOD2- specific monoclonal antibody 6F6. Cells were treated with 10 µM MDP for 3 h as indicated. Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. E) Protein-protein binding assays using <i>in vitro</i> transcribed and translated [35S]-methionine-labeled NOD2 and recombinant GST or GST-TRIM27 (WT, ΔRING or ΔRING+B-Box) bound to glutathione-Sepharose beads. The coomassie-stained gel (bottom) and the autoradiograph (top) of co-precipitated NOD2 are shown.</p

Mapping of the interaction domains in NOD2 and TRIM27.

<p>The depicted NOD2 (A) or TRIM27 (B) deletion constructs (upper panels) were used for the domain mapping. Lysates were subjected to immunoprecipitation using anti-Flag beads and immunoblotted with the indicated antibodies.</p