REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-ERK1 + ERK2 antibody	Abcam	Cat# ab17942
anti-β-actin	Santa Cruz Biotechnology	Cat# I-19
anti-RIPK1	BD-Biosciences	Cat# 610459
anti-β-tubulin	Abcam	Cat# ab6046
anti-β-tubulin-HRP	Abcam	Cat# ab21058
Anti-MAP Kinase, Activated (Diphosphorylated ERK-1&2) antibody	Sigma Aldrich	Clone MAPK-YT, M9692
anti-MLKL	Merck Millipore	Cat# MABC604, Clone 3H1
Anti-phospho-MLKL (Ser345)	Merck Millipore	Cat# MABC1158, clone 7C6.1
Phospho-MEK1/2 (Ser217/221)	Cell Signalling	Cat# 9121
Anti-MEK1/2	Cell Signalling	Cat# 9122
anti-RIPK3	Santa Cruz	Cat# sc-374639
anti-RSK1/2/3	Cell Signalling	Cat# 9355, clone 32D7
anti-phspho-p90-RSK (Ser380)	Cell Signalling	Cat# 9341
anti-GSK3β	Cell Signalling	Cat# 12456, clone D5C5Z-XP
anti-phospho-GSK-3α/β	Cell Signalling	Cat# 9331
anti-mouse Alexa Fluor 488	Thermo Fischer	Cat# A-10667
HRP-conjugated anti-rabbit IgG	Santa Cruz Biotechnology	Cat# sc-2004
HRP-conjugated anti-mouse IgG	Santa Cruz Biotechnology	Cat# sc-2005
HRP-conjugated anti-goat IgG	Santa Cruz Biotechnology	Cat# sc-2020
HRP-conjugated anti-rat- IgG	GE Healthcare	Cat# NA935
Chemicals, peptides, and recombinant proteins
Recombinant mouse TNFα	In house production	N/A
Anti-hFas ligand	Upstate	Cat# 05-201Clone CH11
Phorbol 12-myristate 13-acetate (PMA)	Sigma Aldrich	Cat# P8139
U0126	Sigma Aldrich	Cat# U120
PD184352	Sigma Aldrich	Cat# PZ0181
AZ628	Sigma Aldrich	Cat# SML0785
BI-D1878	Sigma Aldrich	Cat# 559286
Forskoline	Tocris	Cat# 1099/10
SCH772984	Selleckchem	Cat# S7101
Selumetinib (AZD6244)	Selleckchem	Cat# S1008
Trametinib (GSK1120212)	Selleckchem	Cat# S2673
Necrostatin-1S (Nec1S)	Laboratory of Medicinal Chemistry; University of Antwerp	Cat# UAMC-02197
Propidium Iodide	Sigma Aldrich	Cat# P-4170
Sytox Green	Thermo Fischer Scientific	Cat# S7020
Doxycycline Hyclate (Dox)	Sigma Aldrich	Cat# D9891
Rneasy Mini Kit	Qiagen	Cat# 74104
Qiashredder	Qiagen	Cat# 79654
DNA-free™ DNA Removal Kit	Invitrogen	Cat# AM1906
SuperScript® III First-Strand Synthesis System for RT-PCR	Invitrogen	Cat# 18080-51
qPCR MasterMix Plus for SYBR® Green I No ROX	Eurogentec	Cat# RT-SN2X-03+NR
Ac-DEVD-AMC	Pepta Nova	Cat# 3171-V
Bio-Gel P-6 Micro Bio-Spin chromatography columns	Biorad	Cat# 732-6222
plasmid purification columns	Qiagen	Cat# 28106, 28706, and 27106
PCR colony screening kit	5Prime	Cat# MasterTaq Kit
Q5 DNA polymerase	NEB	Cat# M0491
Taq DNA polymerase	NEB	Cat# M0273
Klenow fragment	NEB	Cat# M0212
T4 Polynucleotide Kinase (PNK)	NEB	Cat# M0201
T4 DNA ligase	NEB	Cat# M0202
dNTPs	NEB	Cat# N0447
ATP	NEB	Cat# P0756
Restriction Enzymes, endonucleases	NEB	N/A
four-fragment Multisite Gateway Pro Plus system	Life Technologies	Cat# 12537-100
EDTA-free Complete protease inhibitor cocktail tablets	Roche Diagnostics Belgium	Cat# 11873580001
phosphatase inhibitor cocktail tablets	Roche Diagnostics Belgium	Cat# 04906837001, PhosSTOP
Luminata Classico Western HRP Chemiluminescence Detection Reagents	Millipore	Cat# WBLUC0500
Experimental models: Cell lines
HeLa	ECACC	Cat# 93021013
HEK 293T	ATCC	Cat# CRL-11268
L929sAhFas	Generated in our unit	N/A
HT-29	ATCC	Cat# ATCC-HTB-38
inducible mRIPK3-CT26	Generated in our unit	Aaes et al. (2016)
Fadd-/-Jurkat	Generated in our unit	N/A
Mlkl-/- L929sAhFas	Generated in our unit	N/A
inducible mMLKL(S345D)-L929sAhFas	Generated in our unit	N/A
Recombinant DNA
AKAR4.0	This study	N/A
EKAR4.0	This study	N/A
AKAR3-EV	Kindly provided by the Michiyuki Matsuda laboratory (Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan)	Komatsu, N., Aoki, K., Yamada, M., Yukinaga, H., Fujita, Y., Kamioka, Y., and Matsuda, M. (2011). Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656.
AKAR-EV (T>A)	This study	N/A
EKAR-EV	Kindly provided by the Michiyuki Matsuda laboratory (Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan)	Komatsu, N., Aoki, K., Yamada, M., Yukinaga, H., Fujita, Y., Kamioka, Y., and Matsuda, M. (2011). Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656.
EKAR-EV (T>A)	This study	N/A
EKAR2G-WT	A gift from Olivier Pertz	Addgene plasmid # 39813 ; http://n2t.net/addgene:39813 ; RRID:Addgene_39813
EKAR2G-105/WT	A gift from Olivier Pertz	Addgene plasmid # 39818 ; http://n2t.net/addgene:39818 ; RRID:Addgene_39818
EKAR-EV-VV	Previously developed	Vandame, P., Spriet, C., Riquet, F., Trinel, D., Cailliau-Maggio, K., and Bodart, J.-F. (2014). Optimization of ERK activity biosensors for both ratiometric and lifetime FRET measurements. Sensors (Basel). 14, 1140–1154.
pDG2-flag-rtTA3-puro-mRIPK3	Previously generated in our unit	Aaes et al. (2016)
pDG2-flag-rtTA3-puro-mMLKL(S345D)	Generated in our unit	N/A
Software and algorithms
Statistical analyses	GraphPad Prism	PRISM 8 software, RRID: SCR_002798
qPCR analysis	Biogazelle	qBasePLUS, RRID: SCR_003370
FRET analysis (short experiment)	IGOR Pro - Wavemetrics	Ratioscope_8.31, DOI:10.25493/5G5V-HBC
FRET analysis (long experiment)	Matlab, MathWorks	CellTraFIC code, RRID: SCR_001622
CellTraFIC (Cellular Tracking in FRET Image aCquisition)	APP Paris	IDDN.FR.001.230009.000.S.P. 2017.000.21000
CellTraFIC (Cellular Tracking in FRET Image aCquisition)	GitHub	https://github.com/ayleray/CellTraFIC
Cell Death Analysis	Fiji	RRID: SCR_002285
Immunofluorescence quantification	Cell Profiler	RRID: SCR_007358