KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| NADK2 Antibody | Abcam | Cat# ab181028 Lot# GR164717-1 |
| Anti-rabbit-secondary antibody | Cell Signaling Technology | Cat#7074 Lot# 29 |
| HRP-conjugated anti-mouse-secondary antibody | Cell Signaling Technology | Cat#7076 Lot# 34 |
| β-actin Antibody | Sigma-Aldrich | A5316 Lot# 059M4770V |
| Bacterial and virus strains | ||
| NEB Stable Competent E. coli | NEB | C3040 |
| BL21(DE3) Competent E. coli | NEB | C2527 |
| Chemicals, peptides, and recombinant proteins | ||
| DMSO | Sigma-Aldrich | D2650-100ML |
| Proline | Sigma-Aldrich | P5607-25G |
| 13C5 glutamine | Sigma-Aldrich | 605166-500MG |
| NEB-10-beta/Stable Outgrowth Media | NEB | B9035S |
| Crystal violet | Sigma-Aldrich | C6158-50G |
| Trypab Blue Soloution | Sigma-Aldrich | T8154-100ML |
| L-glutamine | Corning | MT25005Cl |
| Puromycin dichloride | Santa Cruz | sc-108071A |
| Polyethylenimine (PEI) | Polysciences | 23966-2 |
| Opti-MEM media | Thermo Fisher | 31985062 |
| Glutamine-free DMEM | Gibco | 11960-044 |
| SuperSignal™ West Femto Chemiluminescent | Thermo Fisher scientific | PI34096 |
| SuperSignal™ West Pico PLUS | Thermo Fisher scientific | PI34580 |
| DMEM | Corning CellGro | 10-017-CV |
| Fetal bovine serum (FBS) | R&D Systems a biotech brand | S11150 |
| Fetal Bovine Serum dialyzed | R&D Systems a biotech brand | S12850 |
| Microcystin-LR | Enzo Life Sciences | NC9580520 |
| Protease inhibitor cocktail | Sigma-Aldrich | P8340-5ML |
| Acrylamide Protogel 30% | Fisher Scientific | EC-890 |
| Bio-Rad Braford | Bio-Rad | 5000006 |
| Low melting Agarose | Lonza | 50101 |
| Tryptone | United States Biological Life sciences | 91079-40-2 |
| Yeast extract | Euromedex | UY2010 |
| Sodium Chloride | Euromedex | 1112-A |
| Tris Ultra Pure | Euromedex | 26-128-3094-B |
| Imidazole | Millipore | 288-32-4 |
| DTT | Euromedex | EU0006 |
| IPTG | Euromedex | EU0018 |
| InstantBlue® Coomassie Protein Stain | abcam | ab119211 |
| Complete™, EDTA-free Protease Inhibitor cocktail | Roche® Life Science Products | 05056489001 |
| Ampicillin | Euromedex | EU0400 |
| Chloramphenicol | Euromedex | 3886 |
| Tris Ultra Pure | Euromedex | 26-128-3094-B |
| NaCl | Euromedex | 1112-A |
| Sodium citrate tribasic dihydrate | Sigma-Aldrich | S4641 |
| Manganese(II) chloride tetrahydrate | Sigma-Aldrich | M8054 |
| Magnesium chloride hexahydrate | Sigma-Aldrich | M2670 |
| Calcium chloride | Sigma-Aldrich | C1016 |
| Cobalt(II) chloride | Sigma-Aldrich | 60818 |
| Cadmium chloride | Sigma-Aldrich | 202908 |
| Nickel(II) chloride hexahydrate | Sigma-Aldrich | 72247 |
| Copper(II) chloride dihydrate | Sigma-Aldrich | C6641 |
| Zinc chloride | Sigma-Aldrich | 31650-M |
| Barium chloride | Sigma-Aldrich | 202738 |
| Cadmium chloride | Sigma-Aldrich | 202908 |
| Iron(III) chloride | Sigma-Aldrich | F7134 |
| Aluminum chloride hexahydrate | Sigma-Aldrich | A0718 |
| NAD+ | Roche | 10127973001 |
| NADP+ disodium salt | Roche | 10128058001 |
| Adenosine 5′-triphosphate disodium salt hydrate | Sigma-Aldrich | A7699 |
| Sodium polyphosphate | Supelco | 1.06529 |
| D-Glucose 6-phosphate sodium salt | Sigma-Aldrich | G7879 |
| Glucose-6-phosphate Dehydrogenase from baker’s yeast (S. cerevisiae) | Sigma-Aldrich | G7877 |
| Critical commercial assays | ||
| HiSpeed Plasmid Maxi Kit (25) | Qiagen | 12663 |
| His TALON™ Buffer Set | TAKARA | 635651 |
| His TALON™ Gravity Column | TAKARA | 635655 |
| ZymoPURETM I Plasmid Midi Prep Kit | Zymo Research | D4201-B |
| Deposited data | ||
| NADK2 crystal structures | PDB IDs/accession numbers | 7R4J, 7R4K, 7R4L, 7R4M |
| A study by Mary and Soflaee et al., unprocessed files | Mendeley Data | https://doi.org/10.17632/8ybbnh7g3p.1 |
| Experimental models: Cell lines | ||
| HEK293E | Carol Mackintosh (University of Dundee) | N/A |
| ΔNADK2 HEK293E | Tran et al., 2021 | N/A |
| ΔNADK2 HA-Mito | Tran et al., 2021 | N/A |
| ΔNADK2 HEK293E +EV | This study | N/A |
| ΔNADK2 HEK293E +NADK2 | This study | N/A |
| ΔNADK2 HEK293E +Δ78-114-Flag | This study | N/A |
| ΔNADK2 HEK293E +Δ231-266 | This study | N/A |
| ΔNADK2 HEK293E +Δ325-365 | This study | N/A |
| ΔNADK2 HEK293E +S188A | This study | N/A |
| ΔNADK2 HEK293E +T229A | This study | N/A |
| ΔNADK2 HEK293E +S289A | This study | N/A |
| ΔNADK2 HEK293E +S376A | This study | N/A |
| ΔNADK2 HEK293E +K76Q | This study | N/A |
| ΔNADK2 HEK293E +K98Q | This study | N/A |
| ΔNADK2 HEK293E +K104Q | This study | N/A |
| ΔNADK2 HEK293E +K170Q | This study | N/A |
| ΔNADK2 HEK293E +K176Q | This study | N/A |
| ΔNADK2 HEK293E +K304Q | This study | N/A |
| ΔNADK2 HEK293E +K317Q | This study | N/A |
| ΔNADK2 HEK293E +K339Q | This study | N/A |
| ΔNADK2 HEK293E +K355Q | This study | N/A |
| ΔNADK2 HEK293E +K397Q | This study | N/A |
| ΔNADK2 HEK293E +V334R | This study | N/A |
| ΔNADK2 HEK293E +R378Q | This study | N/A |
| ΔNADK2 HEK293E +V334R-R378Q | This study | N/A |
| Recombinant DNA | ||
| pLenti III NADK2-Flag | Tran et al., 2021 | N/A |
| pLenti III NADK2-NADK2-Flag | This study | N/A |
| pLenti III NADK2-Δ78-114-Flag | This study | N/A |
| pLenti III NADK2-Δ231-266 | This study | N/A |
| pLenti III NADK2-Δ325-365 | This study | N/A |
| pLenti III NADK2-S188A-Flag | This study | N/A |
| pLenti III NADK2-T229A-Flag | This study | N/A |
| pLenti III NADK2-S289A-Flag | This study | N/A |
| pLenti III NADK2-S376A-Flag | This study | N/A |
| pLenti III NADK2-K76Q-Flag | This study | N/A |
| pLenti III NADK2-K98Q-Flag | This study | N/A |
| pLenti III NADK2-K104Q-Flag | This study | N/A |
| pLenti III NADK2-K170Q-Flag | This study | N/A |
| pLenti III NADK2-K176Q-Flag | This study | N/A |
| pLenti III NADK2-K304Q-Flag | This study | N/A |
| pLenti III NADK2-K317Q-Flag | This study | N/A |
| pLenti III NADK2-K339Q-Flag | This study | N/A |
| pLenti III NADK2-K355Q-Flag | This study | N/A |
| pLenti III NADK2-K397Q-Flag | This study | N/A |
| pLenti III NADK2-V334R-Flag | This study | N/A |
| pLenti III NADK2-R378Q-Flag | This study | N/A |
| pLenti III NADK2-V334R-R378Q-Flag | This study | N/A |
| pET15b Δ1-60 NADK2 | This study | N/A |
| pET15b Δ1-60 NADK2-Δ78-114 | This study | N/A |
| pET15b Δ1-60 NADK2-Δ231-266 | This study | N/A |
| pET15b Δ1-60 NADK2-Δ325-365 | This study | N/A |
| pET15b Δ1-60 NADK2-S188A | This study | N/A |
| pET15b Δ1-60 NADK2-K76Q | This study | N/A |
| pET15b Δ1-60 NADK2-K304Q | This study | N/A |
| pET15b Δ1-60 NADK2-V334R | This study | N/A |
| pET15b Δ1-60 NADK2-R378Q | This study | N/A |
| pET15b Δ1-60 NADK2-V334R/R378Q | This study | N/A |
| Software and algorithms | ||
| Discover MP 2.4.2 | Refeyn | https://www.refeyn.com/software-release-updates |
| Prism | Prism | https://www.graphpad.com/scientific-software/prism/ |
| TraceFinder 5.1 | ThermoScientific | https://www.thermofisher.com/us/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/lc-ms-data-acquisition-software/tracefinder-software.html |
| ASTRA 6.1 | Waytt Technology | https://www.wyatt.com/products/software/astra.html |
| GraFit | Erithacus Software Ltd | v. 7.0.3 |
| Other | ||
| Mass Photometry TwoMP | Refeyn | N/A |
| SpectraMax iD3- Microplate reader | Molecular devices | N/A |
| Nitrocellulose membrane 0.2 μm | GE Healthcare | 10600001 |
| Blotting paper, 703 | VWR North America | 732-0591 |
| LUNA-II™ Automated Cell Counter | LUNA-II™ | N/A |
| Celigo image cytometer-4 Channel | Nexcelom Bioscience,UK | N/A |
| QExactive HF-X Orbitrap Mass Spectrometer | ThermoScientific | N/A |
| Orbitrap Exploris 480 Mass Spectromete | ThermoScientific | N/A |
| Amicon Ultra-15, PLTK, membrane Ultracel-PL, 30 kDa | Millipore | UFC903024 |
| Complete™ His-Tag Purification Column | Roche® Life Science Products | 06781535001 |
| Bolt™ 4 à 12 %, Bis-Tris | Invitrogen™ | NW04125BOX |
| CLARIOstar plate reader BMG LABTECH | BMG LABTECH | UV-Star® Half area |
| 96-well plates | Greiner | 655087 |