A toolkit for studying cell surface shedding of diverse transmembrane receptors
- University of Minnesota, United States
Figures
Figure 1 with 5 supplements
SEA-like domains cooperate with adjacent domains to behave as proteolytic switches.
(A) Schematic of Synthetic Notch Assay for Proteolytic Switches (SNAPS). Cells co-expressing Flag-Notch-X-Gal4 chimeras, where X is a putative proteolysis region of another receptor, and luciferase …
Figure 1—figure supplement 1
SEA domain chimeras without signaling activity.
Luciferase reporter gene activity profile of Notch chimera constructs co-cultured with MS5 cells or MS5 cells stably transfected with DLL4, including treatment with BB-94 metalloprotease and GSI …
Figure 1—figure supplement 2
ELISA in the presence of BB-94.
Cell surface ELISA performed with DMSO (negative control) or BB-94 (pan-metalloproteinase inhibitor). Data is normalized to the signal of DMSO condition. Error bars represent the SEM of triplicate …
Figure 1—figure supplement 3
Titration of DNA used in co-culture assay.
Luciferase reporter gene activity profile of Notch in comparison to the Notch chimera constructs with SEA/SEA-like domains (A and C) or diverse receptors (B and D) co-cultured with MS5 cells or MS5 …
Figure 1—figure supplement 4
Plated ligand assay.
Luciferase reporter gene activity profile of Notch in comparison to the Notch chimera constructs plated in wells with no ligand or 20 μg/ml recombinant DLL4 ectodomain. Data shown are triplicate …
Figure 1—figure supplement 5
Shedding of diverse receptors detected by SNAPS.
(A) Chimera constructs. Protein domains are color coded and labeled. (B) Luciferase reporter gene activity profile of Notch chimera constructs co-cultured with MS5 cells or MS5 cells stably …
Figure 2
Dystroglycan containing intact proteolytic switch domain is protected from MMP cleavage.
(A) Chimera constructs used to test MMP sensitivity of Notch-Dag chimeras (B) Luciferase reporter gene activity of Notch-Dag chimeras containing intact proteolytic switch and truncated switch with …
Figure 3
SNAPS detects HER2 shedding and shedding modulation by Herceptin.
(A) Chimera constructs for Notch and HER2 (human epidermal growth factor receptor 2. Protein domains are color coded and labeled. (B,C) SNAPS assay measuring effect of Herceptin on basal signaling …
Figure 4 with 1 supplement
SNAPS reveals that E-cadherin proteolysis is a likely mechanism for DECMA-1 disruption of cell-cell adhesion.
(A) Scheme of Notch-E-cadherin (CADH1) chimera in which cadherin repeats 4 and 5 replace the Notch NRR. (B) SNAPS assay on Notch-CADH1 chimera (top) and a construct with 10 amino acids containing …
Figure 4—figure supplement 1
DECMA-1 additional quantification.
(A) EC50 calculation. The dose-dependent proteolytic activity enhancement of DECMA-1 was calculated by first subtracting the IgG control luciferase signal from the DECMA-1 signal at each …
Additional files
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Supplementary file 1
Amino acid sequences for all proteolysis domains used in Notch-X chimeras.
- https://doi.org/10.7554/eLife.46983.012
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Transparent reporting form
- https://doi.org/10.7554/eLife.46983.013
