KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Anti-p-Akt T308 | Cell Signaling | Cat# 2965; RRID# AB_2255933 |
| Anti-pan-Akt | Cell Signaling | Cat# 2920; RRID# AB_1147620 |
| Anti-p-Akt S473 | Cell signaling | Cat# 4060; RRID# AB_2315049 |
| Anti-p-Erk1/2 T202/Y204 | Cell signaling | Cat# 4377; RRID# AB_331775 |
| Anti-Erk1/2 | Cell signaling | Cat# 4696; RRID# AB_390780 |
| Anti-GAPDH | Cell signaling | Cat# 5174; RRID# AB_10622025 |
| Ras10 | Millipore | Cat# 05-516; RRID# AB_2121151 |
| Alexa Fluor 680 goat anti-mouse IgG | Invitrogen | Cat# A21058; RRID# AB_2535724 |
| Alexa Fluor Plus 800 goat anti-rabbit IgG | Invitrogen | Cat# A32735; RRID# AB_2633284 |
| Bacterial and Virus Strains | ||
| Escherichia coli (E. Coli) BL21(DE3) competent cells | New England Biolabs | Cat#C2527I |
| E. Coli One Shot® Top10 competent cells | Invitrogen | Cat#C404010 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| KRAS(1-166) G13D | This paper | N/A |
| KRAS(1-166) wildtype | This paper | N/A |
| HRAS(1-166) G13D | This paper | N/A |
| HRAS(1-166) wildtype | This paper | N/A |
| NRAS(1-166) G13D | This paper | N/A |
| RAF1-RBD (51-131) | This paper | N/A |
| GTP [γ-32P] | Perkin Elmer | BLU004Z250UC |
| 50% Polyethylene glycol 3350 | Hampton Research | HR2-527 |
| PEG/Ion #28 / PEG/Ion HT C4 185mL | Hampton Research | HR2-922-28 |
| Reagent 9 Crystal screen | Hampton Research | HR2-110 |
| Reagent 41 Crystal screen | Hampton Research | HR2-110 |
| Hematoxylin | Sigma | MHS16 |
| Eosin | Thermo Fisher | 6766008 |
| Permount | Fisher Scientific | SP15-100 |
| Mg2+ Lysis/Wash buffer (MLB) | Millipore | 20-168 |
| Glutathione Sepharose 4B beads | GE Healthcare | 17075601 |
| cOmplete™ protease inhibitor cocktail | Roche | 11697498001 |
| Phosphatase inhibitor cocktails 2 | Sigma | P5726 |
| Phosphatase inhibitor cocktails 2 | Sigma | P0044 |
| Benzamidine | Sigma | 434760 |
| Leupeptin | Fisher scientific | L2884 |
| Antipain | Fisher scientific | 10791 |
| Odyssey® phosphate buffered saline blocking buffer | LICOR | 927-40000 |
| MK2206 (Akt inhibitor) | ChemieTek | CT-MK2206 |
| SCH772984 (Erk inhibitor) | ChemieTek | CT-SCH772 |
| GIBCO Advanced DMEM/F-12 media | Thermo Fisher | 12634028 |
| GIBCO HEPES (1M) | Thermo Fisher | 15630080 |
| GIBCO GlutaMAX™ Supplement | Thermo Fisher | 35050061 |
| GIBCO B-27™ Supplement (50X) | Thermo Fisher | 17504044 |
| GIBCO N-2™ Supplement (100X) | Thermo Fisher | 17502048 |
| N-Acetyl-L-cysteine | Sigma | A9165 |
| Nicotinamide | Sigma | N3376 |
| Human [Leu15]-Gastrin I | Sigma | G9145 |
| A83-01 | Sigma | SML0788 |
| SB 202190 | Sigma | S7067 |
| Recombinant human EGF | Thermo Fisher | PHG0311 |
| PGE2 | Sigma | P5640 |
| Primocin | InvivoGen | ant-pm-1 |
| Matrigel® Matrix | Corning | 356235 |
| GIBCO FBS, Lot 1883434 | Thermo Fisher | 16000-044 |
| Corning™ DMEM with L-Glutamine, 4.5g/L glucose and sodium pyruvate | Fisher Scientific | MT10013CV |
| Antibiotic Antimycotic Solution (100 ×), Stabilized | Sigma | A5955 |
| TrypLE Express Enzyme (1X), no phenol red | Thermo Fisher | 12604021 |
| Corning™ Dispase | Fisher Scientific | 354235 |
| Critical Commercial Assays | ||
| Crystal Screen | Hampton Research | HR2-110 |
| Bicinchoninic acid assay (BCA) | Pierce | 23225 |
| CellTiter-Glo® 3D Cell Viability Assay | Promega | G9683 |
| Deposited Data | ||
| HRAS G13D GppNHp—C121 | This paper | PDB: 6E6C |
| HRAS G13D GppNHp— P212121 | This paper | PDB: 6E6P |
| HRAS G13D GDP | This paper | PDB: 6DZH |
| KRAS G13D GppNHp | This paper | PDB: 6E6F |
| KRAS G13D GDP | This paper | PDB: 6E6G |
| NRA SG13D GppNHp | This paper | PDB: 6E6H |
| Experimental Models: Cell Lines | ||
| Fabp1-Cre colon organoids | This paper | N/A |
| Fabp1-Cre; KRasLSL-G12D/+ colon organoids | This paper | N/A |
| Fabp1-Cre; KRasLSL-G13D/+ colon organoids | This paper | N/A |
| L-WRN | ATCC | CRL-3276 |
| Experimental Models: Organisms/Strains | ||
| Mouse: KRasLSL-G13D/+: N/A | Laboratory of Matthew Steensma |
N/A |
| Mouse: Fabp1-Cre: FVB/N-Tg(Fabp1-Cre)1Jig/Nci | NCI Mouse Repository | 01XD8 |
| Mouse: KRasLSL-G12D: B6.129-Krastm4Tyj/Nci | NCI Mouse Repository | 01XJ6 |
| Mouse: B6C3F1/J: female donors | Jackson Laboratories | 100010 |
| Oligonucleotides | ||
| Primers for mutagenesis can be found in Table S2 | This paper | N/A |
| Primers for genotyping can be found in Table S2 | This paper | N/A |
| Primers and oligonucleotides for CRISPR can be found in Table S2 | This paper | N/A |
| Recombinant DNA | ||
| Human: HRAS(1-166) cDNA | This paper—three stop codons are present after | N/A |
| Human: KRAS(1-166) cDNA | Genewiz—Full-length KRAS4B with 3 codons after residue 166 | https://www.genewiz.com/en |
| Human: NRAS(1-166) cDNA | Genewiz—Full-length NRAS with 3 codons after residue 166 | https://www.genewiz.com/en |
| Human: Raf1 GST-RBD 1-149 | Addgene | Plasmid #: 13338 |
| Human: Raf1-RBD 51-131 | Mattos lab | N/A |
| Software and Algorithms | ||
| PHENIX | Adams et al., 2010 | https://www.phenix-online.org/ |
| Coot | Emsley et al., 2010 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ |
| HKL-3000R | Minor et al., 2006 | https://www.hkl-xray.com/hkl-3000 |
| TopSpin™ ver 3.5 | Bruker Corporation | https://www.bruker.com |
| PRISM ver 5-7 | GraphPad Software | https://www.graphpad.com/scientific-software/prism/ |
| DynaFit4 | Kuzmic, 2009 | http://www.biokin.com/index.html |
| MikroWin 300 SL | HIDEX 300SL | http://hidex.com |
| Other | ||
| Corning® 384-well Black/Clear Bottom Ultra-Low Attachment Microplate | Corning | 4588 |