REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Monoclonal Anti-β-Actin mouse antibody (1:4000)	Sigma-Aldrich	Cat# A2228; RRID: AB_476697
Anti-H7 v2 Rabbit (1:1000) polyclonal antibody for HYlight detection	Generated by Genscript	RRID: SCR_002891
Anti-histidine tag Mouse monoclonal antibody (1:1000)	Bio-rad	Cat# MCA1396G; RRID: AB_322084
Anti-Mouse IgG (Fc specific)–Peroxidase antibody produced in goat (1:10000)	Sigma-Aldrich	Cat# A0168; RRID: AB_257867
Goat Anti-Rabbit IgG Antibody, HRP-conjugate (1:5000)	Sigma-Aldrich	Cat# 12–348; RRID: AB_390191
Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:500)	Thermofisher Scientific	Cat# A-21206; RRID:AB_2535792
Bacterial and virus strains
XL1-Blue E. coli strain	Messens lab repository, originally Agilent	–
Chemicals, peptides, and recombinant proteins
William’s E Medium, GlutaMAX™ Supplement	Thermofisher Scientific	Cat# 32551087
Fetal Bovine Serum, qualified, USDA-approved regions	Thermofisher Scientific	Cat# 10437028
Penicillin-Streptomycin (10,000 U/mL)	Thermofisher Scientific	Cat# 15140122
Gibco™ HEPES (1M)	Fisher Scientific	Cat# 15-630-080
Hoechst 33342	Thermofisher Scientific	Cat# 62249
Propidium iodide, ≥94.0% (HPLC)	Merck	Cat# P4170-100MG
DMEM, low glucose, GlutaMAX™ Supplement, pyruvate	Thermofisher Scientific	Cat# 21885108
Seahorse XF DMEM Medium pH 7.4	Agilent Technologies	Cat# 103575-100
Seahorse XF 200 mM glutamine solution	Agilent Technologies	Cat# 103579-100
Seahorse XF 1.0 M glucose solution	Agilent Technologies	Cat# 103577-100
Oligomycin A	MedChem Express	Cat# HY-16589
2-deoxy-D-glucose	Sigma-Aldrich	Cat# D6134
Dexamethasone	Sigma-Aldrich	Cat# D4902
Glucagon	Sigma-Aldrich	Cat# G2044
5-chloro-2-(N-(2,5-dichlorobenzenesulfonamide))-benzoxazole (FBPase-1 Inhibitor)	Santa Cruz Biotechnology	Cat# sc-221608
Seahorse XFp Cell Culture Miniplate	Agilent Technologies	Cat# 103025-100
Sodium L-lactate,∼98%	Sigma-Aldrich	Cat# L7022-5G
Sodium pyruvate, ReagentPlus®, ≥99%	Sigma-Aldrich	Cat# P2256-100G
Cell Lysis Buffer (10×)	Cell Signaling Technology	Cat# 9803S
Halt™ Protease and Phosphatase Inhibitor Cocktails, Thermo Scientific, Halt Protease and Phosphatase Inhibitor Cocktail	Life Technologies	Cat# 78442
Trizma (Base)	Sigma-Aldrich	Cat# T6066
tris(2-carboxyethyl)phosphine (TCEP)	Carl Roth	Cat# HN95.2
Ampicillin Sodium	Duchefa	Cat# A0104
LB Broth High salt	Duchefa	Cat# L1704
Isopropyl-β-D-Thiogalactopyranoside, Dioxane free	Inalco	Cat# 1758-1400
Trizma® hydrochloride	Sigma-Aldrich	Cat# T5941
PMSF Protease Inhibitor	Thermofisher Scientific	Cat# 36978
Leupeptin Protease Inhibitor	Thermofisher Scientific	Cat# 78435
DNase I, RNase-free (1 U/μL)	Thermofisher Scientific	Cat# EN0521
Dithiothreitol (DTT)	Duchefa	Cat# D1309
Imidazole	Millipore	Cat# 288-32-4
D-Fructose 1,6-bisphosphate trisodium salt hydrate	Sigma-Aldrich	Cat# F6803
Bio-Rad Protein Assay Dye Reagent Concentrate	Bio-rad	Cat# 5000006
Carbohydrates Kit	Sigma-Aldrich	Cat# CAR10
TCA Cycle Metabolite Library	Sigma-Aldrich	Cat# ML0010
Guanosine 5′-triphosphate sodium salt hydrate	Sigma-Aldrich	Cat# G8877
Guanosine 5′-diphosphate sodium salt	Sigma-Aldrich	Cat# G7127
α-D-Glucose 1-phosphate dipotassium salt hydrate	Sigma-Aldrich	Cat# G6750
6-Phosphogluconic acid trisodium salt	Sigma-Aldrich	Cat# P7877
D-(−)-3-Phosphoglyceric acid disodium salt	Sigma-Aldrich	Cat# P8877
2,3-Diphospho-D-glyceric acid pentasodium salt	Sigma-Aldrich	Cat# D5764
Sodium L-lactate	Sigma-Aldrich	Cat# L7022
Dihydroxyacetone phosphate lithium salt	Sigma-Aldrich	Cat# 37442
Phospho(enol)pyruvic acid monopotassium salt	Sigma-Aldrich	Cat# P7127
D-Fructose 6-phosphate dipotassium salt	Sigma-Aldrich	Cat# F1502
DL-Glyceraldehyde 3-phosphate	Sigma-Aldrich	Cat# 39705
Adenosine 5′-triphosphate disodium salt solution	Sigma-Aldrich	Cat# A6559
β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate	Sigma-Aldrich	Cat# N1630
β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate	Sigma-Aldrich	Cat# N8129
β-Nicotinamide adenine dinucleotide hydrate	Sigma-Aldrich	Cat# N7004
Glucose 6-phosphate	Roche	Cat# 10127647001
Saponin	Sigma-Aldrich	Cat# 47036
Fructose 1,6-bisphosphate (U-1 ³C₆)	Buchem	Cat# CLM-8962
(2E)-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one, 3PO (inhibitor of glucose metabolism)	Sigma-Aldrich	Cat# SML1343
Dmog,≥98% (hplc) (Dimethyloxalylglycine)	Sigma-Aldrich	Cat# D3695
William's Medium E w/o L-Glutamine, w/o Glucose, w/o Phenol red	Genaxxon Bioscience	Cat# C4318.0500TK
Critical commercial assays
Pierce™ BCA Protein Assay Kits	Thermofisher Scientific	Cat# 23225
PureYield™ Plasmid Miniprep System	Promega	Cat# A1222
Wizard® SV Gel and PCR Clean-Up System	Promega	Cat# A9282
Deposited data
Fructose 1,6-bisphosphate quantification	Metabolomics Workbench (NMDR)	NMDR: https://doi.org/10.21228/M8WB25
HYlight Mass Spectrometry	ProteomeXchange	PRIDE: PXD051879
Experimental models: Cell lines
HepG2 hepatoma cell line (Male)	ATCC	Cat# HB-8065; RRID: CVCL_0027
Huh6 hepatoma cell line (Male)	–	RRID: CVCL_4381
HLE hepatoma cell line (Male)	–	RRID: CVCL_1281
Experimental models: Organisms/strains
C57BL6N Mice	Janvier-Labs	Cat# C57BL/6NRj Mouse RRID: IMSR_RJ:C57BL-6NRJ
Oligonucleotides
Fwd PCS2+/PQE30 EcoRI/BamHI HYlight Human E. coli primer: GGCACGTAGAATTCGGATCCATGGGCAGCAAAGACGTGCT	This paper.	N/A
Rev PCS2+/PQE30 XhoI/HindIII HYlight Human E. coli primer: GGCACGTACTCGAGAAGCTTTTAGCCGCTTTCATCGCGCA	This paper.	N/A
Forward Sequencing HYlight primer: GATCTACCAGAACCGTCAGATCCG	This paper.	N/A
Reverse Sequencing HYlight primer: GATGTTCTAGAGTCCGGAAGCTG	This paper.	N/A
pCS2+ vector Forward Sequencing primer: TGTTCTTTTTGCAGGATCCCATCG	This paper.	N/A
pCS2+ vector Reverse Sequencing primer: TCACTGCATTCTAGTTGTGGTTTG	This paper.	N/A
pQE-30 vector Forward Sequencing primer: AGGAGAAATTAACTATGAGAGG	This paper.	N/A
pQE-30 vector Reverse Sequencing plasmid: CCAGATGGAGTTCTGAGGTC	This paper.	N/A
Recombinant DNA
HYlight codon-optimized E. coli/H. sapiens	This paper, generated by Twist Bioscience	https://www.twistbioscience.com/
pQE-30 vector	Qiagen	https://www.qiagen.com/ko-us
pCS2+ vector	pCS2+HyPer7 was a gift from Vsevolod Belousov	Addgene plasmid # 136466; http://n2t.net/addgene:136466; RRID:Addgene_136466
HYlight codon-optimized M. musculus	Provided by John N. Koberstein	Koberstein et al.5
Software and algorithms
Snapgene	Snapgene	https://www.snapgene.com/; RRID: SCR_015052
TubeSeq Services Eurofins Genomics	Eurofins Genomics	https://eurofinsgenomics.eu/en/custom-dna-sequencing/eurofins-services/tubeseq-services/
NIS-Elements	Nikon	https://www.microscope.healthcare.nikon.com/products/software/nis-elements; RRID: SCR_014329
Fiji	https://doi.org/10.1038/nmeth.2019	https://fiji.sc/; RRID: SCR_002285
GraphPad Prism	GraphPad Software	https://www.graphpad.com/; RRID: SCR_002798
SoftMax Pro	Molecular Devices	https://es.moleculardevices.com/products/microplate-readers/acquisition-and-analysis-software/softmax-pro-software; RRID: SCR_014240
Amersham ImageQuant 800	Cytiva	https://www.cytivalifesciences.com/en/be/shop/protein-analysis/molecular-imaging-for-proteins/imaging-systems/amersham-imagequant-800-systems-p-11546
Xcalibur software	Thermo Scientific	Cat# OPTON-30965; RRID: SCR_014593
ColabFold	Milot Mirdita	https://github.com/sokrypton/ColabFold; RRID: SCR_025453
AlphaFold2	Google DeepMind	https://deepmind.google/technologies/alphafold/; RRID: SCR_025454
AlphaFill	Netherlands Cancer Institute	https://alphafill.eu/
PyMOL	Schrödinger	https://www.pymol.org/; RRID: SCR_000305
Proteome Discoverer Software	Thermofisher Scientific	https://www.thermofisher.com/be/en/home/industrial/mass spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis/proteome-discoverer-software.html; RRID: SCR_014477
FragPipe 20.0	Alexey Nesvizhskii’s proteome bioinformatics group	https://fragpipe.nesvilab.org/; RRID: SCR_022864
Other
μ-Slide 8 Well high ibiTreat cell plate	Ibidi	Cat# 80806
Lipofectamine™ 3000 Transfection Reagent	Thermofisher Scientific	Cat# L3000001
Nb207 Agarose Beads	Kindly provided by Jan Steyaert Laboratory	https://doi.org/10.1038/s41592-020-01001-6
Cytiva Disposable PD-10 Columns	Cytiva	Cat# GE17-0851-01
4–15% Mini-PROTEAN® TGX™ Precast Protein Gels	Bio-rad	Cat# 4561086
InstantBlue® Coomassie Protein Stain (ISB1L)	Abcam	Cat# ab119211
Filtropur S 0.45 μm	Sarstedt	Cat# 83.1826
Ni2+-Sepharose High-Performance Agarose Beads	Cytiva	Cat# 17526802
Vivaspin® 20 Centrifugal Concentrator Polyethersulfone 10 kDa	Sartorius	Cat# VS2001
HiLoad 16/600 Superdex 200 pg	Cytiva	Cat# 28989335
MICROPLATE, 96 WELL, UV-STAR®, COC, F-BOTTOM	Greiner Bio-one	Cat# 655809
6-well plates, TC treated	VWR	Cat# 7341599
U(H)PLC Column ACQUITY HSS T3 C18, 100 Å, 1.8 μm, 2.1 × 150 mm	Waters	Cat# WT186003540
Acclaim™ PepMap™ 100 C18 HPLC Columns	Thermofisher Scientific	Cat# 164946
Acclaim PepMap RSLC, 0.075 × 250 mm	Thermofisher Scientific	Cat# 164569
Lab Vision™ Ultra V Block	Thermofisher Scientific	Cat# TA-060-UB
VECTASHIELD® Antifade Mounting Medium with DAPI	VectorLabs	Cat# H-1200