REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Goat polyclonal anti-Actin (I-19) HRP	Santa Cruz Biotechnology	Cat#sc1616 HRP; RRID: N/A
Mouse monoclonal anti-CYLD (E10)	Santa Cruz Biotechnology	Cat#sc-74435; RRID: AB_1122022
Mouse monoclonal anti-ubiquitin (P4D1)	Santa Cruz Biotechnology	Cat#sc-8017; RRID: AB_2315523
Goat anti-biotin	Cell Signaling Technology	Cat#7075; RRID: AB_330923
Rabbit polyclonal anti-OTULIN	Cell Signaling Technology	Cat#14127; RRID: AB_2576213
Mouse monoclonal anti-IκBα	Cell Signaling Technology	Cat#4814; RRID: AB_390781
Mouse monoclonal anti-phospho-IκBα (Ser32/36) (5A5)	Cell Signaling Technology	Cat#9246; RRID: AB_2151442
Rabbit polyclonal anti-OTUB1	Bethyl Laboratories	Cat#A302-917A; RRID: AB_10663033
Rabbit polyclonal anti-USP5/IsoT	Bethyl Laboratories	Cat#A301-542A; RRID: AB_1040028
Mouse monoclonal anti-FLAG M2	Sigma-Aldrich	Cat#F3165; RRID: AB_259529
Sheep polyclonal anti-HOIL-1/RBCK1	MRC PPU Reagents and Services	Cat#S105D; RRID: N/A
Rabbit polyclonal anti-HOIP/RNF31	Abcam	Cat#ab85294; RRID: AB_1925400
Rabbit polyclonal anti-SHARPIN	Proteintech	Cat#14626-1-AP; RRID: AB_2187734
Rabbit monoclonal anti-M1-polyUb	Millipore	Cat#MABS19; RRID:AB_2576212
Human monoclonal anti-M1-polyUb	Genentech	Matsumoto et al.; RRID: N/A
Rat anti-HA (3F1)	Core facility monoclonal antibodies Helmholtz Zentrum München	RRID: N/A
Bacterial Strains
E. coli BL21-CodonPlus (DE3)-RIPL	Agilent Technologies	Cat#230280
One Shot TOP10 Chemically Competent E. coli	Thermo Fisher Scientific	Cat#C404003
Chemicals, Peptides, and Recombinant Proteins
Recombinant human TNF alpha	biomol	Cat#50435.50
Recombinant M1-linked tetraUb chains	Enzo Life Sciences	Cat#BML-UW0785-0100
Recombinant human diUb chains (K48-linked)	R&D Systems	Cat#UC-200-100
Recombinant human His6-CYLD Isoform 1	R&D Systems	Cat#E-556-050
Recombinant human USP21cat	Laboratory of Titia Sixma	(Faesen et al., 2011)
Recombinant human GST-YOD1	Laboratory of Daniel Krappmann	(Schimmack et al., 2017)
Deconjugating Enzyme Set	R&D Systems	Cat#K-E10B
UbiCREST Deubiquitinase Enzyme Set	R&D Systems	Cat#K-400
Biotin-Ahx-Ub-PA	UbiQ	Cat#UbiQ-076
Fmoc protected amino acids	ChemImpex
PyBOP	ChemImpex	Cat#02276, CAS 128625-52-5
HBTU	ChemImpex	Cat#02011 CAS 94790-37-1
D-Biotin	ChemImpex	Cat#00033, CAS 58-85-5
1,1,1,3,3,3-Hexafluoroisopropanol	ChemImpex	Cat#00080, CAS 920-66-1
Fmoc-Gly-TentaGel Trt R resin (0.18 mmol/g)	Rapp	Cat#RA1213
N-Methyl-2-Pyrrolidone (peptide grade)	Biosolve	Cat#13563304, CAS 872-50-4
Acetonitrile (HPLC grade)	Biosolve	Cat#01201304, CAS 75-05-8
Dichloromethane (S/amylene, peptide grade)	Biosolve	Cat#13793302, CAS 75-09-2
N,N-diisopropylethylamine (peptide grade)	Biosolve	Cat#04153301, CAS 7087-68-5
Water (HPLC grade)	Biosolve	Cat#23210605, CAS 7732-18-5
Water (LCMS grade)	Biosolve	Cat#23217802, CAS 7732-18-5
Piperidine (peptide grade)	Biosolve	Cat#16183301, CAS 110-89-4
Diethyl ether (AR grade)	Biosolve	Cat#05280502, CAS 60-29-7
n-Pentane (AR grade)	Biosolve	Cat#16050502, CAS 109-66-0
Dimethylsulfoxide (AR)	Biosolve	Cat#04470501, CAS 67-68-5
Trifluoroacetic acid (peptide grade)	Biosolve	Cat#20233332, CAS 76-05-1
Formic acid (99%, LCMS grade)	Biosolve	Cat#6914143, CAS 64-18-6
N,N-Dimethylformamide (peptide grade)	Biosolce	Cat#04193301, CAS 68-12-2
Triisopropylsilane (98%)	Sigma Aldrich	Cat#233781, CAS 6485-79-6
Phenol (BioXtra, ≥99.5% GC)	Sigma Aldrich	Cat#P5566, CAS 108-95-2
Methyl 3-mercaptopropionate (98%)	Sigma Aldrich	Cat#108987, CAS 2935-90-2
4-Mercaptophenylacetic acid (97%)	Sigma Aldrich	Cat#653152, CAS 39161-84-7
HOBt	Sigma Aldrich	Cat#54802, CAS 123333-53-9
O-mesitylenesulfonylhydroxylamine	(Bernardes et al., 2008)	CAS 36016-40-7
PR-619, DUB Inhibitor V	Merck Chemicals	Cat#662141; CAS 21645-32-1
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail	Roche	Cat#11836170001
Pierce High Capacitiy Streptavidin Agarose	Thermo Fisher Scientific	Cat#20357
Strep-Tactin Sepharose 50% suspension	IBA	Cat#2-1201-010
Protino® Ni-NTA Agarose	MACHEREY-NAGEL	Cat# 745400.100
Roti-Load 1, 4x conc.	Carl Roth	Cat#K929.1
E.coli recombinant Apyrase	New England Biolabs	Cat#M0398L
Sequence Grade Modified Trypsin	Promega	Cat#V5111
Critical Commercial Assays
Pierce Silver Stain Kit	Life Technologies	Cat#24612
Deposited Data
Structure OTULIN-bio-UbG76Dha-Ub complex	This paper	PDB: 5OE7
Structure OTULIN OTU domain (C129A) in complex with M1-linked diubiquitin	(Keusekotten et al., 2013)	PDB:3ZNZ
Structure zCYLD USP domain (C596S) in complex with M1-linked diubiquitin	Sato et al., 2015	PDB: 3WXE
Mass spectrometry data	ProteomeXchange	PXD006868
Experimental Models: Cell Lines
HEK293 cells	DSMZ	RRID: CVCL_0045
Jurkat T cells	N/A	N/A
Recombinant DNA
pASK-IBA3(+)	IBA	Cat#2-1402-000
pASK-IBA3 OTULINcat-Strep-tag II WT (cat: residues 80-352)	This paper	N/A
pASK-IBA3 OTULINcat-Strep-tag II C129A	This paper	N/A
pASK-IBA3 OTULINcat-Strep-tag II W96A	This paper	N/A
pASK-IBA3 His10-Ub-Ub	This paper	N/A
pASK-IBA3 His10-UbG76A-Ub	This paper	N/A
pASK-IBA3 His10-UbG76C-UbΔG76	This paper	N/A
pEF FLAG mock	Laboratory of Daniel Krappmann	(Scharschmidt et al., 2004)
pEF FLAG-OTULIN WT	This paper	N/A
pEF FLAG-OTULIN C129A	This paper	N/A
pcDNA3.1 (+)	Laboratory of Daniel Krappmann	(Muller-Rischart et al., 2013)
pcDNA3.1 HOIP-HA	Laboratory of Daniel Krappmann	(Muller-Rischart et al., 2013)
pcDNA3.1 HOIL-1-HA	Laboratory of Daniel Krappmann	(Muller-Rischart et al., 2013)
pcDNA3.1 HA-SHARPIN	Laboratory of Daniel Krappmann	(Muller-Rischart et al., 2013)
pEF FLAG-OTUB1	Laboratory of Kamyar Hadian	N/A
pEF FLAG-CYLD	Laboratory of Daniel Krappmann	N/A
pEF FLAG-A20	Laboratory of Daniel Krappmann	(Duwel et al., 2009)
pEF FLAG-YOD1	Laboratory of Daniel Krappmann	(Schimmack et al., 2017)
pEF4 FLAG-UCHL3	Laboratory of Daniel Krappmann	N/A
Software and Algorithms
xia2	(Winter et al., 2013)	http://www.ccp4.ac.uk/
AIMLESS	(Evans and Murshudov, 2013)	http://www.ccp4.ac.uk/
Phaser	(McCoy, 2007)	http://www.ccp4.ac.uk/
COOT	(Emsley et al., 2010)	http://www.ccp4.ac.uk/
Refmac5	(Murshudov et al., 2011)	http://www.ccp4.ac.uk/
PyMOL	PyMOL	http://www.pymol.org
MaxQuant Software (version 1.5.2)		http://www.coxdocs.org/doku.php?id=maxquant:start
Perseus Software (version 1.5.5.3)		http://www.coxdocs.org/doku.php?id=perseus:start
ImageJ	NIH	https://imagej.nih.gov/ij/
Other
DMEM (high glucose, L-glutamine)	Life Technologies	Cat#11965092
RPMI 1640 Medium (L-glutamine)	Life Technologies	Cat#21875034
SOLA HRP SPE Cartridge	Thermo Scientific	Cat#60109-001