Reagent or resource	Source	Identifier
Antibodies
Goat polyclonal anti‐MyD88	R and D Systems	Cat# AF3109; RRID: AB_2146703
Rabbit polyclonal anti‐TRAF6	Enzo	Cat# ADI‐AAP‐426; RRID: AB_10619296
Mouse monoclonal anti‐RBCK1 (HOIL1)	Santa Cruz Biotechnology	Cat# sc‐393754; RRID: NA
Rabbit polyclonal anti‐GFP	ChromoTek	Cat# PABG1; RRID: AB_2749857
Mouse monoclonal anti‐RFP	ChromoTek	Cat# 6g6; RRID: AB_2631395
Mouse monoclonal anti‐GAPDH (6C5)	Thermo Fisher Scientific	Cat# AM4300; RRID: AB_2536381
Mouse monoclonal anti‐tubulin	Abcam	Cat# ab7291; RRID: AB_2241126
Rabbit monoclonal anti‐NF‐κB p65	Cell Signaling Technology	Cat# 8242; RRID: AB_10859369
Anti‐phospho‐IKKα/β	Cell Signaling Technology	Cat# 2697; RRID: AB_2079382
Anti‐phospho‐p65	Cell Signaling Technology	Cat# 3033; RRID: AB_331284
Human monoclonal antilinear (M1‐Ub) polyubiquitin (1F11/3F5/Y102L)	Genentech (Matsumoto et al, 2012)	NA
Human monoclonal anti‐K63‐Ub linked polyubiquitin chain	Genentech (Newton et al, 2008)	NA
Camelid sdAb FluoTag®‐X4 anti‐GFP‐Atto488	NanoTag Biotechnologies GmbH	Cat# N0304‐At488
Goat anti‐rabbit conjugated to Alexa Fluor 647	Invitrogen	Cat# A21246
Goat anti‐human conjugated to Alexa Fluor 647	Invitrogen	Cat# A21445
Nano‐secondary AF568	Chromotek	Cat# srbAF568‐1‐100
Bacterial and virus strains
BL21 Rosetta	Novagen	Cat# 70954
NEB Stable	NEB	Cat# C3040
TOP10	Thermo	Cat# C404003
Chemicals, peptides, and recombinant proteins
His10‐Halo‐Tencon‐SpycatcherV2‐IL‐1β	Deliz‐Aguirre et al (2021)
Puromycin dihydrochloride	Thermo	Cat# A1113803
Blasticidin	InvivoGen	Cat# ant‐bl‐05
Mouse IL‐1β	Peprotech	Cat# 211‐11B
JF646‐HaloTag ligand	Luke Lavis, HHMI
Critical commercial assays
MycoAlert® Mycoplasma Detection Kit	Lonza	Cat# LT07‐218
QuickExtract DNA Extraction Solution	Lucigen	Cat# SS000035‐D2
Mouse IL‐2 DuoSet ELISA	R&D Systems	DY402
Experimental models: cell lines
Mouse: EL4.NOB‐1	ECACC	87020408
Mouse: EL4 MyD88‐GFP	Deliz‐Aguirre et al (2021)
Mouse: EL4 mScarlet‐TRAF6/MyD88‐GFP	This paper
Mouse: EL4 mScarlet‐HOIL1/MyD88‐GFP	This paper
sgRNA sequences
MyD88 GFP knock‐in gRNA1: CCTGCCCTGAAGATGACCCT	Deliz‐Aguirre et al (2021)
TRAF6_mScarlet‐i knock‐in gRNA: GTAAACTGTGAGAACAGCTGC	This paper
HOIL1_mScarlet‐i knock‐in gRNA: GTCTTCTCGTCCATCTGGCC	This paper
Recombinant DNA
pMK	Thermo	GeneArt vector
pX330‐U6‐Chimeric_BB‐CBh‐hSpCas9	Addgene	Plasmid #42230
pMK_MyD88‐GFP_HDRtemp	Deliz‐Aguirre et al (2021)	NA
pX330‐MyD88‐gRNA	Deliz‐Aguirre et al (2021)	NA
pMK_TRAF6‐BlastR‐T2A‐mScarlet‐i_HDRtemp	This paper	NA
pX330‐TRAF6‐gRNA	This paper	NA
pMK_HOIL1‐BlastR‐T2A‐mScarlet‐i_HDRtemp	This paper	NA
pX330‐HOIL1‐gRNA	This paper	NA
Software and algorithms
Fiji	NIH	https://fiji.sc/
MATLAB	MathWorks	https://www.mathworks.com/
R	CRAN	https://cran.r‐project.org/
ggplot2	tidyverse	https://ggplot2.tidyverse.org/
Fiji/MATLAB/R scripts from image analysis	This study	https://github.com/MJ‐Taylor‐Lab/Analysis‐for‐Ub‐paper