REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-Actin, mouse monoclonal	Merck Millipore	Cat# MAB1501; RRID: AB_2223041
Anti-cIAP1, rat monoclonal	Enzo Life Sciences	Cat# ALX-803-335; RRID: AB_2227905
Anti-CYLD, mouse monoclonal	Santa Cruz Biotechnology	Cat# sc-74435; RRID: AB_1122022
Anti-pS418-CYLD, rabbit polyclonal	Cell Signaling Technology	Cat# 4500
Anti-ERK1/2, rabbit monoclonal	Cell Signaling Technology	Cat# 4695; RRID: AB_390779
Anti-phospho-ERK1/2, rabbit monoclonal	Cell Signaling Technology	Cat# 4370; RRID: AB_2315112
Anti-HOIL-1, mouse monoclonal	Santa Cruz Biotechnology	Cat# sc-365523; RRID: AB_10841591
Anti-HOIP, sheep polyclonal	R&D systems	Cat# AF8039; RRID: AB_2714038
Anti-IκBα, rabbit polyclonal	Cell Signaling Technology	Cat# 9242; RRID: AB_331623
Anti-IRAK1, rabbit polyclonal	Santa Cruz Biotechnology	Cat# sc-7883; RRID: AB_2233753
Anti-JNK, rabbit monoclonal	Cell Signaling Technology	Cat# 9258; RRID: AB_2141027
Anti-phospho-JNK, rabbit monoclonal	Cell Signaling Technology	Cat# 9251; RRID: AB_331659
Anti-OTULIN, rabbit polyclonal	Abcam	Cat# ab151117; RRID: AB_2728115
Anti-p38, rabbit polyclonal	Cell Signaling Technology	Cat# 9212; RRID: AB_330713
Anti-phospho-p38, rabbit monoclonal	Cell Signaling Technology	Cat# 4511; RRID: AB_2139682
Anti-RelA/p65, rabbit monoclonal	Cell Signaling Technology	Cat# 8242; RRID: AB_10859369
Anti-pS536-RelA/p65, rabbit monoclonal	Cell Signaling Technology	Cat# 3033; RRID: AB_331284
Anti-RIPK1, rabbit monoclonal	Cell Signaling Technology	Cat# 3493; RRID: AB_2305314
Anti-RIPK2, rabbit polyclonal	Santa Cruz Biotechnology	Cat# sc-22763; RRID: AB_2300888
Anti-SHARPIN, rabbit polyclonal	Proteintech	Cat# 14626-1-AP; RRID: AB_2187734
Anti-TNFR1, rabbit monoclonal	Cell Signaling Technology	Cat# 3736; RRID: AB_2241018
Anti-ubiquitin, mouse monoclonal	Imgenex	Cat# IMG-5021; RRID: AB_317568
Anti-ubiquitin (P4D1 clone), mouse monoclonal	Cell Signaling Technology	Cat# 3936; RRID: AB_331292
Anti-Lys63-linked ubiquitin, rabbit monoclonal	Merck Millipore	Cat# 05-1308; RRID: AB_1587580
Anti-Met1-linked ubiquitin, rabbit monoclonal	Merck Millipore	Cat# MABS199; RRID: AB_2576212
Anti-mouse-HRP, goat	Agilent (previously Dako)	Cat# P0447; RRID: AB_2617137
Anti-rat-HRP, goat	ThermoFisher	Cat# 31470; RRID: AB_228356
Anti-rabbit-HRP, goat	Bio-Rad	Cat# 1706515; RRID: AB_11125142
Anti-sheep-HRP, donkey	R&D systems	Cat# HAF016; RRID: AB_562591
Anti-CXCL8 (IL8)-APC, mouse	BioLegend	Cat# 511410; RRID: AB_893464
Anti-FLAG M2 affinity gel, mouse monoclonal	Sigma-Aldrich (now Merck Millipore)	Cat# A2220; RRID: AB_10063035
Bacterial and virus strains
Rosetta II DE3	Novagen	Cat# 71400
DH10Bac	ThermoFisher	Cat# 10361012
Chemicals, peptides, and recombinant proteins
L18-MDP	Invivogen	Cat# tlrl-lmdp
TNF	Peprotech	Cat# 300-01A
Biotin-TNF	R&D systems	Cat# BT210
FLAG-TNF	Enzo life systems	Cat# ALX-522-008-C050
IL-1β	Cell signaling Technologies	Cat# 2022
Brefeldin A	BioLegend	Cat# 420601
Monensin	BioLegend	Cat# 420701
ICC fixation buffer	BioLegend	Cat# 420801
N-Ethylmaleimide (NEM)	Sigma Aldrich	Cat# E3876
cOmplete protease inhibitors	Roche	Cat# 04693159001
cOmplete Protease Inhibitor tablets	Sigma-Aldrich	Cat# 11873580001
PhosSTOP	Roche	Cat# 04906845001
lambda protein phosphatase	New England Biolabs and Gift from David Barford	Cat# P0773
3C Protease	Elliott et al., 2016; PMID: 27591049	N/A
SUMO Protease	Elliott et al., 2016; PMID: 27591049	N/A
Insect-XPRESS™ Protein-free Insect Cell Medium	Lonza	Cat# BELN12-730Q
DNaseI	Sigma-Aldrich	Cat# DN25-100mg
Lysozyme	Sigma-Aldrich	Cat# L6876-5G
IPTG	VWR	Cat# 437144N
Tris-(2-carboxyethyl)phosphine (TCEP)	Fluorochem	Cat# M02624-10G
Ubiquitin	Elliott et al., 2016 PMID: 27591049	N/A
15N ammonium chloride	Sigma-Aldrich	Cat# 299251-25G
13C glucose	Sigma-Aldrich	Cat# 389374-10G
Critical commercial assays
RevertAid RT Reverse transcriptase	Thermo Fisher Scientific	Cat# K1691
RiboLock RNase Inhibitor	Thermo Fisher Scientific	Cat# EO0381
RNAeasy mini kit	QIAGEN	Cat# 74104
Rnase-free Dnase set	QIAGEN	Cat# 79254
SYBR select Master Mix	Applied Biosystems	Cat# 4472908
PNGase F	New England Biolabs	Cat# P0704
Deposited data
CYLD CAP-Gly3 (467-565): Ub complex	This study	PDB: 7OWC
CYLD CAP-Gly3 (467-552): Ub complex	This study	PDB: 7OWD
CYLD phosphorylation sites by Mass spectrometry	This study	PRIDE: PXD026791
Experimental models: Cell lines
U2OS/FlpIn/Trex/HA-NOD2 (U2OS/NOD2)	Fiil et al., 2013; PMID: 23806334	N/A
U2OS/NOD2 CYLD KO (9E-8 clone)	Elliott et al., 2016; PMID: 27591049	N/A
U2OS/NOD2 CYLD KO + CYLDWT	This study	N/A
U2OS/NOD2 CYLD KO + CYLDL475P	This study	N/A
U2OS/NOD2 CYLD KO + CYLDS418A	This study	N/A
U2OS/NOD2 CYLD KO + CYLDS568A	This study	N/A
U2OS/NOD2 CYLD KO + CYLDS418A, S568A	This study	N/A
U2OS/NOD2 CYLD KO + CYLDC601A	This study	N/A
HCT-116	Cummins et al., 2004; PMID: 15126334	N/A
THP1	ATCC	Cat# TIB-202
U937	ATCC	Cat# CRL-1593.2
Sf9	ThermoFisher	Cat# 11496015
Oligonucleotides
Listed in Table S2
Recombinant DNA
pBabe-puro	Morgenstern and Land, 1990; PMID: 2194165	RRID: Addgene_1764
pBabe-puro-CYLDWT	This study	N/A
pBabe-puro-CYLDL475P	This study	N/A
pBabe-puro-CYLDC601A	This study	N/A
pBabe-puro-CYLDS418A	This study	N/A
pBabe-puro-CYLDS568A	This study	N/A
pBabe-puro-CYLDS418A,S568A	This study	N/A
pTRIP-Puro	Hrdinka et al., 2016; PMID: 26997266	N/A
pTRIP-Puro-shMM	Hrdinka et al., 2016; PMID: 26997266	N/A
pTRIP-Puro-shCYLD	Hrdinka et al., 2016; PMID: 26997266	N/A
pACEBAC-SUMO∗	This study	N/A
pACEBAC-SUMO∗-CYLD 583-956	This study	N/A
pACEBAC-SUMO∗-CYLD 542-956	This study	N/A
pACEBAC-SUMO∗-CYLD 467-956	This study	N/A
pACEBAC-SUMO∗-CYLD 460-956	This study	N/A
pACEBAC-SUMO∗-CYLD 1-956	This study	N/A
pOPINS-CYLD 120-213	This study	N/A
pOPINS-CYLD 226-313	This study	N/A
pOPINS-CYLD 460-582	This study	N/A
pOPINS-CYLD 467-582	This study	N/A
pOPINS-CYLD 467-582L475P	This study	N/A
pOPINS-CYLD 226-313F288D	This study	N/A
pOPINB-NEMO 383-419	This study	N/A
pOPINS-CLIP170 48-359	This study	N/A
pOPINS-CLIP170 48-147	This study	N/A
pOPINS-TBCB 151-244	This study	N/A
pOPINS-TBCE 1-110	This study	N/A
pOPINS-KIF13B 1685-1784	This study	N/A
pOPINS-DCTN1 1-107	This study	N/A
Software and algorithms
GraphPad Prism	GraphPad	https://www.graphpad.com
Adobe Creative Suite	Adobe	https://www.adobe.com
Fiji (ImageJ)	https://www.nature.com/articles/nmeth.2019	https://imagej.net/software/fiji/
FlowJo	BD Biosciences	https://www.bdbiosciences.com/en-us/products/software/flowjo-v10-software
PEAKS Server v1.7	Bioinformatics Solutions Inc.	https://www.bioinfor.com/peaks-online/
Phenix	Adams et al., 2010; PMID: 20124702	https://www.phenix-online.org
COOT	Emsley et al., 2010; PMID: 20383002	https://www2.mrc-lmb.cam.ac.uk/Personal/pemsley/coot
CCP4 7.2	Winn et al., 2011; PMID: 21460441	https://www.ccp4.ac.uk/?page_id=1417
Pymol	Schrödinger, Inc	https://www.pymol.org/2/
Topspin 3.0	BRUKER	https://www.bruker.com/en/products-and-solutions/mr/nmr-software/topspin.html
NMRPipe	Delaglio et al., 1995; PMID: 8520220	https://www.ibbr.umd.edu/nmrpipe/index.html
Analysis 2.4	Vranken et al., 2005; PMID: 15815974	https://www.ccpn.ac.uk/v2-software/software/analysis
Pomana	Shen and Bax, 2015; PMID: 26053889	https://spin.niddk.nih.gov/bax/software/POMONA/
CS-Rosetta 3.7	Shen et al., 2008; PMID: 18326625	https://spin.niddk.nih.gov/bax/software/CSROSETTA/
Haddock 2.2	van Zundert et al., 2016; PMID: 26410586	https://www.bonvinlab.org/software/haddock2.2/
PEAQ Analysis	Malvern Instruments	https://www.malvernpanalytical.com/en/
Other
Ni-NTA	QIAGEN	Cat# 30210
Resource Q	Cytiva Life Sciences	Cat# 17117901
HiLoad Superdex75	Cytiva Life Sciences	Cat# 28989333
HiLoad Superdex 200	Cytiva Life Sciences	Cat# 28989335