Supplementary Materials 
RNF126-mediated ubiquitination of FSP1 affects its subcellular localization and ferroptosis 
Wanqun Xie1*, Jiajia Wang1*, Shuaiwei Tian1*, Heng Zhao1, Liangliang Cao1, Zhuangzhuang Liang1, Jian Yang1, Baocheng Wang1, Yang Zhao1, Qinhua Wang1, Feng Jiang1, Jie Ma1#

SUPPLEMENTAL EXPERIMENTAL PROCEDURES
KEY RESOURCES TABLE
REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies			
Tubulin	Proteintech Group	66031-1-Ig; RRID:AB_11042766	
FLAG	Proteintech Group	20543-1-AP; RRID:AB_11232216	
HA	Proteintech Group	51064-2-AP; RRID:AB_11042321	
MYC	Proteintech Group	60003-2-Ig; RRID:AB_2734122	
GST	Proteintech Group	66001-1-lg	
HIS	Sigma	H1029; RRID:AB_260015	
FSP1	Proteintech Group	20886-1-AP	
RNF126	Proteintech Group	66647-1-Ig	
RNF126	ABclonal	A20015	
GPX4	Sigma	SAB4300725	
xCT	Abmart	AB_2936462	
ACSL4	Abcam	ab155282	
DHODH	Proteintech Group	14877-1-AP	
TOM20	Cell Signaling Technology	42406	
Na,K-ATPase A	Cell Signaling Technology	3010	
4-HNE	Abcam	Ab46545	
Ki-67	Cell Signaling Technology	34330	
Critical Commercial Assays			
CellTiter-Glo® Luminescent Cell Viability Assay	Promega	Cat# G7573	
Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit	Inventbiotech	Cat# SM-005	
Mitochondria Isolation Kit	Thermo Fisher Scientific	Cat# 89874	
TRIzol	Invitrogen	Cat# 15596026	
cDNA Synthesis Kit 	Takara Bio	Cat# 634926	
QIAGEN Plasmid Mini Kit	Qiagen	Cat# 12125	
SYBR Green qPCR Master Mix 	Takara Bio	Cat# RR420L	
Cytotoxicity LDH Assay Kit	Dojindo	Cat# CK12	
GSSG/GSH Quantification Kit II	Dojindo	Cat# G263	
Chemicals			
Lipofectamine 2000 Reagent 	Invitrogen	Cat# 11668019	
BODIPY™ 581/591 C11	Invitrogen	Cat# D3861	
RIPA lysis buffer 	Sigma	Cat# R0278	
protease inhibitor cocktail 	Roche	Cat# 5892970001	
Cycloheximide	R&D Systems	Cat# 0970	
Erastin	Selleck	Cat# S7242	
RSL3	Selleck	Cat# S8155	
FIN56	Selleck	Cat# S8254	
Deferoxamine mesylate	Selleck	Cat# S5742	
Necrostatin-1	Selleck	Cat# S8037	
Z-VAD-FMK	Selleck	Cat# S7023	
N-acetylcysteine	Selleck	Cat# S1623	
Tetrathiomolybdate	Selleck	Cat# E1166	
4-CBA (4-chlorobenzoic acid)	Sigma	Cat# 135585	
iFSP1	Selleck	Cat# S9663	
Ferrostatin-1	Selleck	Cat# S7243	
Sulfasalazine	Selleck	Cat# S1576	
VivoGlo™ Luciferin,In Vivo Grade	Promega	Cat# P1043	
Experimental Models: Cell Lines			
HEK293T	ATCC	CRL-3216; RRID:CVCL_0063	
D283 Med	A gift from Dr. Yu Jie Tang	HTB-185; RRID:CVCL_1155	
D458 Med	A gift from Dr. Yu Jie Tang	RRID:CVCL_1161	
Experimental Models: Organisms/Strains			
nude mouse	Shanghai Slack Laboratory Animal Co., Ltd.	NA	
Recombinant DNA			
pGL3-basic 	Promega	Cat# E1751	
pLVML-Flag	This paper	NA	
pLVML-Myc	This paper	NA	
pLVML-EGFP	This paper	NA	
pLVML-EGFP-FSP1	This paper	NA	
pLVML-EGFP-FSP1(K182R,K193R,K199R,K225R)	This paper	NA	
pLVML-Myc-RNF126	This paper	NA	
pLVML-Flag-FSP1	This paper	NA	
PLVML-Myc-RNF126(△1-40aa)	This paper	NA	
PLVML-Myc- RNF126 (△41-129aa)	This paper	NA	
PLVML-Myc- RNF126 (△130-150aa)	This paper	NA	
PLVML-Myc- RNF126 (△151-219aa)	This paper	NA	
PLVML-Myc- RNF126 (△220-280aa)	This paper	NA	
PLVML-Myc- RNF126 (△281-311aa)	This paper	NA	
PLVML-Flag-FSP1(K43R,K63R,K64R,K74R)	This paper	NA	
PLVML-Flag-FSP1(K88R,K118R,K162R,K168R)	This paper	NA	
PLVML-Flag-FSP1(K182R,K193R,K199R,K225R)	This paper	NA	
PLVML-Flag-FSP1(K230R,K293R,K314R,K322R,K366R)	This paper	NA	
pRK5-HA	This paper	NA	
pRK5-HA-Ub	This paper	NA	
pGBKT7-FSP1	This paper	NA	
pRK5-His-Ub	This paper	NA	
pGEX-4T-1-GST	This paper	NA	
pET28a-His-RNF126	This paper	NA	
Software and Algorithms			
GraphPad	GraphPad	GraphPad	
FlowJo	FlowJo	FlowJo	
ImageJ	ImageJ	ImageJ	
PCR primers			
Primers	Species	Sequences	
FSP1	Human	Forward:AGTAGTGGGGATAGACCTGAAGA
Reverse:CCACCACGATGAACCGTGA	
PTGS2	Human	Forward:CTGGCGCTCAGCCATACAG
Reverse:CGCACTTATACTGGTCAAATCCC	
β-ACTIN	Human	Forward:CGGAACCGCTCATTGCC
Reverse:ACCCACACTGTGCCCATCTA	
Oligos sequences			
Symbols	The targeting sequence (5’-3’)	
Control-sgRNA	CACCGGCACTACCAGAGCTAACTCA	
RNF126-sgRNA#1	CACCGATCTGTCCAAGATGCGAGTC	
RNF126-sgRNA#2	CACCGATAAAACCAGACTCGCATCT	
RNF126-sgRNA#3	CACCGGGGCGACTCACCTCCAACGG	
FSP1-sgRNA#1	CACCGGAATCGGGAGCTCTGCACG	
FSP1-sgRNA#2	CACCGTCCCGATTCCACCGAGACCT	
FSP1-sgRNA#3	CACCGTGAGGCAGTCTCCACCTTGA