1. Volume 46, Issue 6, Pages 746-758 (June 2012)
The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immunity 
Rune Busk Damgaard, Ueli Nachbur, Monica Yabal, Wendy Wei-Lynn Wong, Berthe Katrine Fiil, Mischa Kastirr, Eva Rieser, James Arthur Rickard, Aleksandra Bankovacki, Christian Peschel, Juergen Ruland, Simon Bekker-Jensen, Niels Mailand, Thomas Kaufmann, Andreas Strasser, Henning Walczak, John Silke, Philipp J. Jost, Mads Gyrd-Hansen 
Molecular Cell 
Volume 46, Issue 6, Pages (June 2012)
DOI: /j.molcel
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2. Molecular Cell 2012 46, 746-758DOI: (10.1016/j.molcel.2012.04.014)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1
XIAP Is Required for Signaling in Response to NOD2 Stimulation
(A) Lysates from WT and Xiap−/− BMDMs treated with MDP (10 μg/ml) were examined by immunoblotting.
(B) Relative mRNA levels were measured by semiquantitative RT-PCR of WT or XIAP−/− BMDMs treated for 4 hr with MDP (10 μg/ml). Data represent mean ± SEM of six (WT) or four (Xiap−/−) independent experiments.
(C) The concentration of TNF and IL-6 in supernatants from WT or Xiap−/− BMDMs stimulated with MDP (10 μg/ml). Data represent mean ± SEM of three independent experiments each performed in triplicate. ND indicates not detectable.
(D and E) WT and Xiap−/− mice were injected i.p. with vehicle (v) or MDP (25 mg/kg body weight) 4 hr prior to collection of serum of peripheral blood and liver tissue. Serum cytokines were measured by flow cytometry (D), and relative mRNA levels of liver tissue were measured by semiquantitative RT-PCR (E). n.s., not significant. Replicates as indicated in the figure. The two-tailed Student's t test was used to determine statistical significance. See Figure S1.
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4. Figure 2
XIAP Contributes to NOD2-Mediated Inflammation-Induced Liver Injury In Vivo
(A) BMDMs from WT and Xiap−/− mice were stimulated for the indicated times with ultrapure LPS (50 ng/ml) and/or MDP (10 μg/ml) before measurement of secreted TNF and IL-6 in supernatants. n.s., not significant. Data represent mean ± SEM of three independent experiments each performed in triplicate.
(B) WT and Xiap−/− mice were injected i.p. with PBS (vehicle) or MDP (5 mg/kg body weight) 4 hr prior to i.p. injection of ultrapure LPS (125 ng/kg body weight) along with GalN (1 g/kg body weight). Macroscopic images of livers from WT and Xiap−/− mice treated as indicated and sacrificed 5 hr after injection of LPS/GalN.
(C and D) Hematoxylin and eosin (H&E) staining (C) and quantification of TUNEL staining of liver sections (D) from mice treated as in (B). Images in (C) are representative of three mice per genotype and time point. Scale bars: 100 μm. Quantification of TUNEL-positive cells is shown in (D) from five viewing fields per mouse of three independent mice per time point; error bars indicate SEM.
(E) Serum levels of liver transaminases ALT and AST in mice treated as described in (B). n.s., not significant. Two-tailed Student's t test was used for statistical analysis; replicates as indicated in figure. See Figures S1 and S2.
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5. Figure 3
The Ubiquitin Ligase Activity of XIAP Is Critical for NF-κB Activation and Cytokine Secretion in Response to NOD2 Stimulation
(A) Schematic overview of XIAP variants. BIR, baculovirus IAP repeat; UBA, ubiquitin associated; RING, really interesting new gene.
(B) Ubiquitin conjugates were purified from cells expressing Strep-ubiquitin and HA-tagged XIAPWT or XIAPF495A.
(C and D) NF-κB activity in lysates of cells transfected as indicated and stimulated with MDP (10 μg/ml) for 24 hr (C) or transfected with HA-NOD2 (D). Data represent mean ± SEM of three independent experiments, each performed in triplicate. n.s., not significant.
(E) Relative mRNA levels were measured by semiquantitative RT-PCR of WT, XIAP−/−, and XIAPΔRING BMDMs treated for 4 hr with MDP (10 μg/ml). Data represent mean ± SEM of 3–6 independent experiments.
(F) TNF in supernatants from WT, XIAP−/−, and XIAPΔRING BMDMs that had been stimulated with MDP (10 μg/ml) for the indicated time points. A line connects data points from BMDM cultures isolated from individual mice. Each data point represents the average of triplicate measurements. The two-tailed Student's t test was used to determine statistical significance. See Figure S3.
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6. Figure 4 XIAP Ubiquitylates RIPK2 in Response to Stimulation of NOD2
(A) HA-NOD2 was immunoprecipitated from lysates of HEK293T cells transfected with empty vector or HA-NOD2 vector. Immunoprecipitates were examined for copurified proteins by immunoblotting.
(B) Purification of endogenous ubiquitin conjugates from THP-1 lysates after stimulation with L18-MDP (200 ng/ml) or TNF (10 ng/ml). Purified material was examined for ubiquitylated RIPK1 and RIPK2 proteins by immunoblotting.
(C) THP-1 cells were transfected with siRNA targeting XIAP and were stimulated with L18-MDP. Cell lysates were analyzed as in (B).
(D and E) Cells were left untreated or treated with the IAP antagonist LBW-242 (20 μM) for 15 min before stimulation with L18-MDP (200 ng/ml) (D) or TNF (10 ng/ml) (E). Ubiquitin conjugates were isolated from cell lysates and analyzed as in (B).
(F) Ubiquitin conjugates were purified with StrepTactin-Agarose resin from lysates of cells expressing Strep-Ubiquitin and XIAPWT or XIAPF495A. Purified material was examined for ubiquitylated proteins by immunoblotting.
(G and H) Ubiquitin conjugates were purified with anti-HA-Agarose resin from lysates of cells expressing HA-Ubiquitin variants and XIAP as indicated in the figure. Purified material was examined for ubiquitylated proteins by immunoblotting. See Figures S4 and S5.
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7. Figure 5
XLP-2-Derived Mutations in XIAP Affect Its RING Activity and Impair NOD2 Signaling
(A) Alignment of RING domain and C-terminal region of XIAP-like proteins. Blue and magenta colors indicate Zn-coordinating Cys and His residues, respectively. The green and red labels indicate residues altered in XIAP variants identified in XLP-2 patients. The XLP-2-associated mutations studied here are indicated above the alignment. Species abbreviations are as follows: Hs (Homo sapiens), Mm (Mus musculus), Clf (Canis lupus familiaris), Ss (Sus scrofa), Xl (Xenopus laevis), Dr (Danio rerio), Dm (Drosophila melanogaster).
(B) Cartoon depiction of NMR structure of the RING domain of human XIAP (Protein Data Bank [PDB] ID: 2ECG). The red and green labels indicate the residues mutated in XLP-2 patients.
(C and D) Ubiquitin conjugates were purified with StrepTactin-Agarose resin from lysates of cells expressing Strep-Ubiquitin and XIAP variants as indicated. Purified material was examined by immunoblotting as indicated. Note that lanes 1–3 in (C) are also shown in Figure 3B.
(E–G) NF-κB activity in lysates of cells transfected as indicated and treated with L18-MDP (200 ng/ml) for 24 hr (F). Data represent mean ± SEM of 3–6 independent experiments, each performed in triplicate. The two-tailed Student's t test assuming unequal variance was used to determine statistical significance.
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8. Figure 6 LUBAC Is Recruited to NOD2 by XIAP
(A) Cells expressing HA-NOD2 were untreated or treated with LBW-242 (20 μg/ml) 1 hr prior to lysis and immunoprecipitation of HA-NOD2. Immunoprecipitates were examined by immunoblotting for copurification of components of the NOD2 signaling complex.
(B) HA-NOD2 was immunoprecipitated from lysates of cells expressing HA-NOD2 and FLAG-XIAP (WT or F495A). Immunoprecipitates were examined for copurification of components of the LUBAC complex. Asterisk indicates unspecific band detected by the anti-FLAG antibody.
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9. Figure 7
LUBAC Is Required for Efficient NF-κB Activation and Cytokine Secretion in Response to NOD2 Stimulation
(A) NF-κB activity in lysates of cells depleted for LUBAC subunits as indicated and treated with MDP (10 μg/ml) for 24 hr.
(B) NF-κB activity in lysates of cells with or without stable knockdown of HOIL-1 after transfection with HA-NOD2 or FLAG-XIAPWT vectors.
(C) Relative mRNA levels of Tnf and Il6 in WT or cpdm BMDMs treated with L18-MDP (200 ng/ml). Data represent mean ± SEM of four (WT) or three (cpdm) independent experiments). n.s., not significant.
(D) TNF and IL-6 in supernatants from WT and cpdm BMDMs stimulated with L18-MDP (200 ng/ml). A line connects data points from BMDM cultures isolated from individual mice. Each data point represents the average of triplicate measurements.
(E and F) NF-κB activity in lysates of cells transfected with the expression vectors indicated. Data in (A), (B), (C), (E), and (F) represent mean ± SEM of at least three independent experiments, each performed in triplicate. The two-tailed Student's t test was used to determine statistical significance. See Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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