Analysis of cancer mutations introduced into the Drosophila melanogaster Notch negative regulatory region uncovers a diversity of regulatory outcomes
- School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, United Kingdom
Figures
Figure 1 with 1 supplement
Activation of Notch signal by gain-of-function mutations in the Drosophila Notch negative regulatory region (NRR).
(A) Front and side views of the human NOTCH1 NRR crystal structure, highlighting residues corresponding to four Drosophila gain-of-function mutations: l1N-B (G1572V; dark cyan), 414 (D1577G; green), CC-SS (C1693S and C1696S; yellow), and LGI >AAA (LGI1514–1516AAA; blue). The heterodimerisation (HD) domains (HD-N; pale cyan and HD-C; cyan) are shown as ribbons to illustrate secondary structure and the positions of mutations within the fold, particularly those at the HD-N/HD-C interface. The Lin12/Notch repeats (LNR) repeats are shown in space-filling representation to highlight their surface-exposed positions and facilitate visual distinction from the HD domains. All mutations in this study are uniquely colour-coded for consistency across structural representations and quantitative analyses, including NRE-luciferase assays and protein-level measurements. (B) Enhanced ligand-independent signalling by Notch gain-of-function mutants. NRE-luciferase assay in S2 cells shows that all four gain-of-function Notch mutants exhibit significantly increased basal signalling activity compared to wild-type. (n=6 for CC-SS and LGI-AAA, n=10 for L1NB and 414) (C–E) Ligand (Dl) and Deltex (Dx)-induced Drosophila Notch signal. Wild-type Notch signal can be further activated by Dl and Dx, n=16 (C). Dl- (D) and Dx- (E) induced Notch signal intensity of the four gain-of-function mutants were normalised by the signal from wild-type Notch, n=3 for CC-SS and LGI-AAA, n=5 for L1NB and 414. p<0.05 (*), p<0.01 (**), and p<0.001 (***) by two-tailed t-test, error bars are SEM.
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Figure 1—source data 1
Raw data used for quantifications in panels B-E.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Inducibility of negative regulatory region (NRR) mutants by Dl and Deltex (Dx) expressed as a % of wild-type (WT).
(A, B) Inducibility of Drosophila NRR mutant alleles by Dl (A) and Dx (B) activation, n=3 for CC-SS/LGI-AAA and n=5 for G1572V (l1N-B allele) and D1577G (414 allele). (C) Fold-decrease of 414 allele after Dx expression compared to 414 basal, n=5. (D, E) Inducibility of cancer-associated heterodimerisation domain (HD) and Lin12/Notch repeats (LNR) domain mutants by Dl (D) and Dx (E), n=3 except for E1705P, n=4. (F,G) Inducibility of LNR-C dimer interface mutants in response to Dl (F), and Dx (G), n=5. To calculate relative inducibility, all basal signals were first normalised to 1. The ‘fold difference’ was calculated as induced signal –1 for each mutant and for WT control, and then the mutant value expressed as a percentage of the WT fold difference. Hence 100% indicates inducibility of mutant is same as WT, 0% indicates mutant signal does not change in induced conditions compared to its own basal signal, and negative numbers are cases where the mutant signal is reduced in the presence of Dl or Dx, compared its own basal condition. * indicates p<0.05 compared to WT (A, B, D–G) or compared to 414 basal (C), by two-tailed t-test, error bars are SEM.
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Figure 1—figure supplement 1—source data 1
Raw data used for quantifications in panels A-G.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 1 supplement
Functional analysis of T-cell acute lymphoblastic leukaemia (T-ALL)–derived heterodimerisation domain mutations in Drosophila Notch.
(A) Positions of residues corresponding to seven Drosophila mutations (F1617P; dark magenta, R1626Q; dark blue, H1630P; strong pink, L1632P; soft magenta, R1633P; violet, L1686P; magenta, and E1705P; dark turquoise) in heterodimerisation domain (HD) domain shown on human NOTCH1 NRR crystal structure. The selected residues represent recurrent, independently reported cancer-associated NOTCH1 HD mutations. F1617P, L1632P, and L1686P lie in the hydrophobic core; H1630P and R1633P are surface-exposed; and R1626Q, and E1705P are positioned at the Lin12/Notch repeats (LNR)–HD boundary. (B) Basal Notch activity of the seven HD domain mutants, demonstrating that the only two mutants R1626Q and E1705P exhibit accelerated signal intensity in ligand-independent manner, like T-ALL mutants, n=6 except for E1705P, n=8. (C,D) Notch signal activation of HD domain mutants by ligand (C) and Dx (D), normalised by wild-type Notch activated by ligand and Dx, respectively, n=3 except for E1705P, n=4. (E) Impaired secretory trafficking of Notch HD domain mutants due to ER/Golgi-retention. The localisations of two representative non-signalling HD mutants in conserved residues were investigated (L1632P, L1686P) and compared with the active signalling mutant G1515K. Upper panels: EGFP-tagged wild-type Notch and two HD domain mutants (L1632P and L1686P) were expressed in S2 cells and Notch protein localisation (EGFP, green) was analysed by immunofluorescence. The cells were stained with anti-PDI (for ER, red) and anti-GM130 (for Golgi, blue) antibodies. There was no clear difference between WT and G1515K localisation despite the intense basal signal of G1515K (Figure 3B). Lower panels: higher magnification of the box shown in L1632P image. Scale bar: 5 µm (upper panels) and 1 µm (lower panels). (F) Effect of PEST domain truncation on ligand-independent Notch signal of HD domain mutants. PEST domain in the C-terminal region of Notch was removed from four representative mutants (R1626Q, L1632P, L1686P, and E1705P), and the basal signal was analysed by NRE-luciferase assay, n=4. Only R1626Q and E1705P show T-ALL mutants-like synergistic increase of the signal. p<0.05 (*), p<0.01 (**), and p<0.001 (***) by two-tailed t-test, error bars are SEM.
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Figure 2—source data 1
Raw data used for quantifications in panels B-D, F.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Positions of negative regulatory region (NRR) point mutations used in this study, shown in the context of amino acid conservation.
Alignment of the five domains (LNR-A/B/C and HD-N/C) from different Notch proteins (Drosophila Notch, human NOTCH1–4, mouse NOTCH1, zebrafish Notch1a, and C. elegans LIN-12) was colour-coded: green for hydrophobic residues, red for acidic, violet for basic, yellow for cysteine, orange for serine and threonine, cyan for tyrosine and tryptophan, light purple for asparagine and glutamine, and light yellow for glycine and proline.
Figure 3
Structure-function analysis of the Drosophila Notch Lin12/Notch repeats (LNR) domain via cancer-derived mutational mapping.
(A) Distribution of cancer-associated LNR mutations on the human NOTCH1 negative regulatory region (NRR) structure. Fourteen mutations (E1489V; olive, D1497N; dark green, A1504V; aquamarine, G1515K; blue, T1523M; salmon, N1529E; red pink, E1538R; crimson, D1548N; maroon, E1553P; cyan, R1554C; brown, L1562P; yellow, H1570P; strong yellow, D1573V; dark yellow, and Y1578R; tan) were introduced into Drosophila Notch, based on cancer-associated variants reported in the human NOTCH1 LNR domain. The D1548N mutation, which contributes to a calcium-binding site, is buried within the LNR and is not visible in the figure. (B) Basal Notch activity of the LNR domain mutants, demonstrating that the only two mutants G1515K and E1553P exhibit apparently high signal intensity in a ligand-independent manner, n=6. (C, D) Notch signal activation of the LNR mutants by ligand (C) and Deltex (Dx) (D), normalised by wild-type Notch activated by ligand and Dx, respectively, n=3. (E) Synergistic enhancement of ligand-independent Notch signalling by combined LNR mutations and PEST domain truncation, n=4. The effect of PEST domain truncation on the basal activity of two gain-of-function LNR mutants (G1515K and E1553P) was assessed using the NRE-luciferase assay. The combination led to a synergistic increase in ligand-independent signalling, resembling the behaviour of T-ALL–associated NOTCH1 mutations. p<0.05 (*), p<0.01 (**), and p<0.001 (***) by two-tailed t-test, error bars are SEM.
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Figure 3—source data 1
Raw data used for quantifications in panels B-E.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig3-data1-v1.xlsx
Figure 4
Identification of a novel regulatory hotspot in Lin12/Notch repeats (LNR)-C driving ligand-independent Notch activation.
(A) Human NOTCH1 NRR crystal structure highlighting the positions of three surface-exposed LNR-C mutations (F1563A; amber, Y1566A; pale yellow, and H1570A; orange). (B) Strong increase in basal (ligand-independent) Notch signalling induced by the three LNR-C mutations, n=10. (C, D) Ligand-dependent (C) and Deltex (Dx)-induced (D) activation of the LNR-C mutants, normalised to wild-type Notch activation under the same conditions, n=5. (E) Effect of PEST domain truncation on ligand-independent signalling of the LNR-C mutants, n=4. p<0.05 (*), p<0.01 (**), and p<0.001 (***) by two-tailed t-test, error bars are SEM.
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Figure 4—source data 1
Raw data used for quantifications in panels B-E.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig4-data1-v1.xlsx
Figure 5
Intact NRR–NRR homodimerisation despite Lin12/Notch repeats (LNR)-C interface mutations.
(A,B) Co-immunoprecipitation–based assay for negative regulatory region (NRR)–NRR homodimerisation. (A) Schematic presentation of the assay. EGFP-tagged full-length Notch was immunoprecipitated from S2 cells co-expressing Notch–EGFP and luciferase-tagged Notch LNR domain using an anti-GFP antibody (GFP-trap). Homodimerisation was assessed by measuring co-precipitated luciferase activity. (B) Luciferase signals were normalised to total lysate luciferase activity to quantify binding efficiency. Robust NRR–NRR homodimerisation was observed, and none of the tested interface mutations significantly affected this interaction, n=4. (C,D) Split-luciferase assay to assess the effect of LNR-C mutations on NRR–NRR homodimerisation. (C) Schematic representation of the assay. N-terminal and C-terminal fragments of luciferase were fused to Notch LNR constructs and co-expressed in S2 cells. Homodimerisation was detected as reconstituted luciferase activity. (D) Firefly luciferase signals were normalised to Renilla luciferase activity to quantify dimerisation efficiency, n=5, except for WT + no LNR, n=3. Strong luciferase activity indicated NRR–NRR homodimerisation, and none of the tested LNR-C interface mutations significantly altered this interaction. All constructs include N-terminal signal peptides to ensure proper membrane topology. p<0.05 (*), p<0.01 (**), and p<0.001 (***) by two-tailed t-test, error bars are SEM.
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Figure 5—source data 1
Raw data used for quantifications in panels B, D.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
A novel negative regulatory function of Lin12/Notch repeats (LNR)-C in Notch protein stability.
(A, B) Protein expression levels of Notch cancer mutants in S2 cells. Expression profiles of all cancer-derived Notch mutants used in this study were examined by western blotting (A), and the band intensities of full-length Notch were quantified to compare relative protein levels, n=3 (B). (C, D) Elevated Notch protein levels by LNR-C Interface Mutations. Expression profiles of LNR-C interface mutants and two Drosophila alleles, l1N-B (G1572V) and 414 (D1577G), were examined by western blotting (C), and the band intensities of full-length Notch were quantified to compare relative protein levels, n=4 (D). (E,F) Enhanced stability of Notch LNR-C interface mutants revealed by cycloheximide (CHX) chase assay. (E) S2 cells expressing LNR-C interface mutants (F1563A or H1570A) were treated with 10 µM cycloheximide for the indicated time and assessed by western blotting. (F) Band intensities of full-length Notch were quantified to evaluate protein stability over time, n=4, except for F1563A, n=3. p<0.05 (*), p<0.01 (**), and p<0.001 (***) by two-tailed t-test, error bars are SEM.
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Figure 6—source data 1
Raw data used for panels B, D, F.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig6-data1-v1.xlsx
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Figure 6—source data 2
Original, unlabelled, western blots used for A-F.
For C, D, two different exposures are shown for each blot to allow positions of markers to be visualised.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig6-data2-v1.zip
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Figure 6—source data 3
PDF files containing original labelled western blots used for A-F.
Relevant bands are indicated.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig6-data3-v1.zip
Figure 6—figure supplement 1
Lin12/Notch repeats (LNR)–heterodimerisation domain (HD) interaction mutations do not affect Notch protein stability, as revealed by a cycloheximide (CHX) chase assay.
(A) S2 cells expressing LNR–HD interaction mutants (G1515K and E1705P) or a PEST domain–truncated variant were treated with 10 µM cycloheximide for the indicated times and analysed by western blotting. (B) Band intensities of full-length Notch were quantified to assess protein stability over time. * indicates p<0.05 compared to wild-type (WT) (two-tailed t-test), error bars are SEM, n=3, except for WT, n=7.
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Figure 6—figure supplement 1—source data 1
Raw data used for quantifications panel B.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig6-figsupp1-data1-v1.xlsx
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Figure 6—figure supplement 1—source data 2
Original, unlabelled, western blots used for A, B.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig6-figsupp1-data2-v1.zip
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Figure 6—figure supplement 1—source data 3
PDF file containing original labelled western blots used for A, B.
Relevant bands are indicated.
- https://cdn.elifesciences.org/articles/108812/elife-108812-fig6-figsupp1-data3-v1.zip
Tables
Table 1
List of human NOTCH1 cancer mutations and their Drosophila equivalents used in this study.
Summary of 22 cancer-associated mutations analysed in this study. Drosophila Notch mutations were designed based on reported human NOTCH1 cancer mutations. Both conserved and non-conserved residues were included to examine how structural perturbations at equivalent positions affect signalling activity, independent of sequence conservation. The table lists each Drosophila mutation alongside its corresponding human mutation, structural location, associated cancer types, and relevant references.
| Drosophila | Human NOTCH1 | Location | Cancer types | Reference |
|---|---|---|---|---|
| E1489V | E1456V | LNR-A | Prostate cancer (PCa) | Mateo et al., 2020 |
| D1497N | S1464N | LNR-A | PCa, squamous cell carcinoma (SCC) | Mateo et al., 2020; South et al., 2014 |
| A1504V | A1471V | LNR-A | Ampullary adenocarcinoma | Gingras et al., 2016 |
| G1515K | N1482K | LNR-A/B | Metastatic colorectal cancer (mCRC) | Yaeger et al., 2018 |
| T1523M | T1491M | LNR-B | Merkel cell carcinoma | Starrett et al., 2020 |
| N1529E | K1498E | LNR-B | Skin melanoma | Bi et al., 2017 |
| E1538R | S1507R | LNR-B | Melanoma | Abou Alaiwi et al., 2020 |
| D1548N | D1517N | LNR-B | Head and neck (HN) SCC, T-cell acute lymphoblastic leukaemia (T-ALL) | Neumann et al., 2015; Stransky et al., 2011 |
| E1553P | Q1522P | LNR-B/C | SCC | South et al., 2014 |
| R1554C | R1523C | LNR-B/C | Cervical SCC | Zhang et al., 2016 |
| L1562P | L1531P | LNR-C | Oral (O) SCC | Izumchenko et al., 2015 |
| H1570P | H1539P | LNR-C | OSCC | Izumchenko et al., 2015 |
| D1573V | D1542V | LNR-C | Oesophageal SCC | Zhang et al., 2015 |
| D1577G (414) | D1546G | LNR-C | T-ALL | Asnafi et al., 2009 |
| Y1578R | Q1547R | LNR-C | Mantle cell lymphoma | Zhang et al., 2014 |
| F1617P | L1585P | HD-N | T-ALL | Asnafi et al., 2009; Neumann et al., 2015; Weng et al., 2004 |
| R1626Q | R1594Q | HD-N | T-ALL, SCC, stomach | Durinck et al., 2011; Ito et al., 2019; Zhang et al., 2016 |
| H1630P | R1598P | HD-N | T-ALL | Asnafi et al., 2009; Neumann et al., 2015; Weng et al., 2004 |
| L1632P | L1600P | HD-N | T-ALL, breast | Asnafi et al., 2009; Neumann et al., 2015; Pop et al., 2018; Weng et al., 2004 |
| R1633P | H1601P | HD-N | T-ALL | Matteucci et al., 2010 |
| L1686P | L1678P | HD-C | T-ALL, mCRC, stomach | Asnafi et al., 2009; Liu et al., 2014; Neumann et al., 2015; Weng et al., 2004; Yaeger et al., 2018 |
| E1705P | A1701P | HD-C | T-ALL | Neumann et al., 2015; Weng et al., 2004 |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | Notch (N) | Drosophila melanogaster | https://flybase.org CG3936 | |
| Gene (Drosophila melanogaster) | Deltex (dx) | Drosophila melanogaster | https://flybase.org CG3929 | |
| Gene (Drosophila melanogaster) | Suppressor of deltex (Su(dx)) | Drosophila melanogaster | https://flybase.org CG4244 | |
| Gene (Drosophila melanogaster) | Delta (Dl) | Drosophila melanogaster | https://flybase.org Dl CG3619 | |
| Cell line (Drosophila melanogaster) | S2 | Thermo Fisher Scientific | Cat# R69007 RRID:CVCL_Z232 | |
| Cell line (Drosophila melanogaster) | S2-Mt-Dl | DGRC | Cat# 152 RRID:CVCL_Z993 | |
| Antibody | Mouse monoclonal anti-PDI | Enzo life sciences | 1D3 Cat# ADI-SPA-891 RRID:AB_916902 | IF(1:500), |
| Antibody | Rabbit polyclonal anti-GM130 | Abcam | Cat# ab30637 RRID:AB_732675 | IF(1:500) |
| Antibody | Mouse monoclonal anti-Notch (ICD) | Developmental Studies Hybridoma Bank | Cat# C17.9C6 RRID:AB_528410 | WB 1:10000 |
| Antibody | Rabbit polyclonal anti-β-actin | Proteintech | Cat#:81115–1-RR RRID:AB_2923704 | WB 1:10000 |
| Recombinant DNA reagent | Plasmid | Shimizu et al., 2024 | NRE-firefly luciferase | Notch responsive expression of Firefly Luciferase |
| Recombinant DNA reagent | Plasmid | Shimizu et al., 2024 | pMT-Renilla luciferase | Metallothionein promoter-driven expression of Renilla |
| Recombinant DNA reagent | Plasmid | Shimizu et al., 2024 | pMT–Dx–V5 | Metallothionein promoter-driven expression of Dx-V5 |
| Recombinant DNA reagent | Plasmid | Shimizu et al., 2024 | pMT–Notch–EGFP | Metallothionein promoter-driven expression of Notch |
| Recombinant DNA reagent | Plasmid | This study | pMT-NE1489V-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-ND1497N-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NA1504V-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NG1515K-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NT1523M-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NN1529E-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NE1538R-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-ND1548N-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NE1553P-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NR1554C-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NL1562P-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NF1563A-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NY1566A-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NH1570P-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NH1570A-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NG1572V-GFP | See Materials and Methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-ND1573V-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-ND1577G-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NY1578R-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NF1617P-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NR1626Q-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NH1630P-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NL1632P-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NR1633P-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NL1686P-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NE1705P-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NCC-SS-GFP | C1693S and C1696S See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-N LGI>AAA -GFP | L1514A G1515A I1516A See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NΔPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NG1515K−ΔPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NE1553P−ΔPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NF1563A−ΔPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NH1570A−ΔPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NG1572V−ΔPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-ND1577G−ΔPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NR1626Q- DPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NL1632P−ΔPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NL1686P−ΔPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-NE1705P−ΔPEST-GFP | See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-LNR-Luc | Co-IP assay See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-LNRF1563A-Luc | Co-IP assay See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-LNRH1570A-Luc | Co-IP assay See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-LNR-NLuc | Split Luciferase assay See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-LNRF1563A-NLuc | Split Luciferase assay See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-LNRH1570A-NLuc | Split Luciferase assay See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-LNR-CLuc | Split Luciferase assay See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-LNRF1563A-CLuc | Split Luciferase assay See Materials and methods |
| Recombinant DNA reagent | Plasmid | This study | pMT-LNRH1570A-CLuc | Split Luciferase assay See Materials and methods |
| Commercial assay or kit | Dual-Glo Luciferase Assay System | Promega | Cat# E2940 | |
| Commercial assay or kit | GFP-Trap | Chromo Tek | Cat# gtma RRID:AB_2631358 | |
| Commercial assay or kit | Effectene | QIAGEN | Cat# 301425 | |
| Commercial assay or kit | Q5 DNA Polymerase | New England Biolabs | Cat# M0491 | |
| Commercial assay or kit | NuPAGE Tris-Acetate Mini Protein Gels, 3 to 8% | Thermo Fisher Scientific | Cat# EA0375, EA03752, EA03755 | |
| Chemical compound, drug | Cycloheximide | Sigma-Aldrich | Cat# C7698 |
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Analysis of cancer mutations introduced into the Drosophila melanogaster Notch negative regulatory region uncovers a diversity of regulatory outcomes
eLife 14:RP108812.
https://doi.org/10.7554/eLife.108812.3
