1. Binding of OTULIN to the PUB Domain of HOIP Controls NF-κB Signaling
Veronique Schaeffer, Masato Akutsu, Michael H. Olma, Ligia C. Gomes, Masato Kawasaki, Ivan Dikic 
Molecular Cell 
Volume 54, Issue 3, Pages (May 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 54, 349-361DOI: (10.1016/j.molcel.2014.03.016)
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3. Figure 1 Identification of LUBAC Interaction Partners
(A) SILAC-based mass spectrometric analysis of LUBAC interactome. Immunoprecipitation of C-terminally HA/2×Strep-tagged (-SSH) HOIP stably expressed in HeLa cells is shown. Bait-expressing cells were heavy labeled; cells expressing no bait were light labeled. Significantly enriched proteins are indicated.
(B) Immunoprecipitation of tagged Sharpin, OTULIN wild-type, and catalytic inactive mutant.
(C) Immunoprecipitation of endogenous OTULIN with endogenous HOIP. OTULIN was immunoprecipitated, and samples were immunoblotted and stained for HOIP and OTULIN. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 OTULIN and p97 Bind to a Conserved Pocket on HOIP PUB Domain
(A) Pull-down analysis of immobilized GST-tagged LUBAC components with OTULIN. Ponceau S staining shows GST-HOIP, -HOIL, and -Sharpin.
(B) Pull-down analysis of immobilized GST-HOIP full-length, PUB-, and NZF-domain constructs with OTULIN. Ponceau-S-stained membrane shows GST-tagged proteins.
(C) Superposition of the ribbon model of crystal structures of the HOIP PUB domain (green) and PNGase PUB domain (pink) in complex with p97 peptide (PDB ID 2HPL). p97 peptide (cyan) is shown in ball-and-stick representation.
(D) Surface representation of the HOIP PUB domain, colored by conserved residues of PUB domain.
(E) A close-up view of the conserved pocket of HOIP PUB domain, in the same view as (C). Hydrophobic surface residues are colored in green.
(F) Close-up view of the p97 peptide-binding pocket of the PNGase PUB domain.
(G and H) Surface representation of PUB domain Ψ–Y pocket of PNGase PUB domain and p97 peptide (ball-and-stick representation, cyan) (G) and HOIP PUB domain (H).
(I) Pull-down analysis of immobilized GST-HOIP PUB wild-type and mutants with OTULIN. Ponceau-S-stained membrane shows GST-tagged constructs.
(J) Pull-down analysis of immobilized GST-HOIP PUB wild-type and mutant with HeLa Lysate, Ponceau S staining as in (I). See also Figures S2 and S3.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 OTULIN and p97 Bind HOIP via Conserved Tyrosine-Based Motif
(A) Pull-down analysis of immobilized OTULIN wild-type and fragments thereof with MBP-tagged HOIP ZF. Ponceau-S-stained membrane shows GST-tagged fragments.
(B) A close-up view of the interaction between HOIP PUB domain and OTULIN peptide (Ball-and-stick representation, yellow), in the same view as Figure 2E. Hydrophobic surface residues are colored by green.
(C) Close-up view of the interaction between HOIP PUB domain and p97 peptide (ball-and-stick representation, cyan), in the same view as Figure 2E.
(D and E) Surface representations of HOIP PUB domain Ψ–Y pocket binding to p97 peptide shown in cyan (D) or to OTULIN peptide shown in yellow (E).
(F and G) Cartoon representation of HOIP PUB domain (green) binding to p97 shown in cyan (F) or binding to OTULIN shown in yellow (G). Hydrogen bonds between HOIP PUB domain the central tyrosines are shown as black dashed lines.
(H) Pull-down analysis of immobilized GST-tagged OTULIN wild-type and Tyr56 mutants with MBP-HOIP ZF. Ponceau S staining shows GST-OTULIN constructs.
(I) Immunoprecipitation of HSS-OTULIN wild-type and Y56F mutant transiently overexpressed in HeLa cells. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 OTULIN Is Recruited at the TNF Receptor via Tyr56
(A–C) TNFR purifications after treatment with HisFlag-TNFα (HF-TNFα) for the indicated times and immunoprecipitation using AntiFlag-resin.
(A) The recruitment of endogenous OTULIN and HOIP to the activated TNFR was detected by immunoblot.
(B and C) Cells expressed transiently HSS-OTULIN wild-type or mutant Y56F prior to the addition of HF-TNFα.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 OTULIN Recruitment via Tyr56 Is Important for NF-κB Signaling
(A) Luciferase assay for NF-κB activity in HeLa cells transfected with Sharpin, HOIP, OTULIN wild-type, or the indicated point mutants.
(B) Luciferase assay for TNFα-induced NF-κB activity in HeLa cells knockdown for OTULIN (siOTULIN) and expressing OTULIN wild-type (wt) or OTULIN Y56F mutant.
(C) p65 nuclear translocation in response to TNFα in HeLa cells knockdown for OTULIN (siOTULIN) and expressing OTULIN wt or OTULIN Y56F mutant. Cells were treated with TNFα for the indicated time. ∗ compared to siOTULIN; # compared to siOTULIN + TNFα + OTULINwt.
(D) DUB activity assessment for GST-OTULIN wt and GST-OTULIN Y56F.
(E) Effect of OTULIN in the luciferase assay for NF-κB activity in HeLa cells expressing sharpin and wt or mutant (Y82A/N85E) HOIP.
(F) Effect of OTULIN on p65 nuclear translocation in cells expressing sharpin and wt or mutant (Y82A/N85E) HOIP. Data are represented as mean ±SEM. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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