(A) Logic diagram for single ‘AND’ gate used in first-generation SPARK1 (Kim et al., 2017). Transcription of the reporter gene requires both light (to open the LOV domain) AND a protein-protein …
(A) Schematic of NanoLuc fused to membrane-localized protein β2AR and asLOV2 in LOVTRAP. When asLOV2 is activated by NanoLuc (or blue light), mCherry-Zdk1 dissociates from the C-terminus of asLOV2. …
Excel spreadsheet containing fluorescence intensity values used to generate panels C-D and G-H.
(A) HEK293T cells were transfected as in Figure 2A. Example sequential immunofluorescence images of mCherry-Zdk1 during baseline (Dark), 10 µM furimazine, and blue light for the intracellular …
Excel spreadsheet containing fluorescence intensity and luminescence values used to generate panels B-D.
(A) Schematic of NanoLuc fused to arrestin-TEVp, co-expressed with GPCR-LOV-TEVcs-Gal4 and UAS-Citrine. In the dark and in the absence of the GPCR’s agonist, the TEVcs is caged by the LOV domain and …
Excel spreadsheet containing fluorescence ratio intensity values used to generate panels D, G, and H.
(A) HEK293T cells were transfected with either β2AR SPARK1 (arrestin-TEVp) or SPARK2 (NanoLuc-arrestin-TEVp) and UAS-Citrine. The same transmembrane construct was used in both conditions …
Excel spreadsheet containing fluorescence intensity ratio values used to generate panel B.
(A) Schematic of the general timeline for SPARK2 assays using an orthogonal luciferase reporter such as FLuc. For simplicity, only the luciferases and LOV domain are illustrated (unfilled …
Excel spreadsheet containing luminescence values used to generate panel B.
Excel spreadsheet containing SPARK2 drug screen compounds and mean fold-changes (n = 2 biological replicates) to generate panels C-E.
(A) Schematic for testing whether NanoLuc activation of eLOV is proximity-dependent. NanoLuc was fused either to arrestin-TEVp (left, Complexed), or expressed at the membrane as CD4-NanoLuc (right, …
Excel spreadsheet containing luminescence values used to generate panels B-D.
(A) Schematic of an extracellular NanoLuc fused to oβ2AR-eLOV-TEVcs-Gal4, co-expressed with NanoLuc-arrestin-TEVp and UAS-Citrine. (B) Citrine expression in HEK293T cells infected with lentiviruses …
Excel spreadsheet containing cell count and fluorescence intensity ratio values used to generate panels C and G.
(A) HEK293T cells were infected with lentiviruses as in Figure 5A. Immunofluorescence images of Citrine expression were taken ~24 hr following 10 min of blue light to activate …
Excel spreadsheet containing cell counts and fluorescence intensity ratio values used to generate panels B and D-E.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (HEK293T) | HEK293T | ATCC | Tested negative for mycoplasma. | |
Antibody | Rabbit anti-HA | Cell Signaling Technology | C29F4 | 1:100 |
Antibody | Goat anti-Rabbit AlexaFluor 647 | Life Technologies | A27040 | 1:1000 |
Antibody | Mouse anti-Flag | Sigma | F3165 | 1:300 |
Antibody | Goat anti-Mouse AlexaFluor 568 | Life Technologies | A11004 | 1:1000 |
Recombinant DNA reagent | See Table 1 for all plasmids used or generated in this study. | |||
Commercial assay or kit | Nano-Glo Live Cell Assay System | Promega | 1:100 substrate | |
Commercial assay or kit | Bright-Glo Luciferase Assay System | Promega | 1:1 substrate | |
Software, algorithm | Fiji | Schindelin et al., 2012 | ||
Software, algorithm | CellSegm toolbox | Hodneland et al., 2013 | ||
Software, algorithm | MATLAB R2017a | Mathworks |
Name | Description | Vector-Promoter | Addgene |
---|---|---|---|
P1 | HA-β2AR-NNES-NanoLuc-15aa linker-AsLOV2(V416L) | pAAV-CMV | 125224 |
P2 | gLuc sp-NanoLuc-HA-β2AR-NNES-AsLOV2(V416L) | pAAV-CMV | 125225 |
P3 | mCherry-GSGS linker-Zdk1 | pAAV-CMV | 125226 |
P4 | DRD1-NNES-eLOV-TEVcs-Flag-Gal4-V5 | pAAV-CMV | 125227 |
P5 | AVPR2-NNES-eLOV-TEVcs-Flag-Gal4-V5 | pAAV-CMV | 104844 |
P6 | β2AR-NNES-eLOV-TEVcs-Flag-Gal4-V5 | pAAV-CMV | 104841 |
P7 | NanoLuc-15aa linker-βarrestin2-HA-GS linker-TEVp | pAAV-CMV | 125228 |
P8 | UAS-Citrine | pAAV-UAS | 104839 |
P9 | βarrestin2-HA-GS linker-TEVp | pAAV-CMV | 104845 |
P10 | UAS-FLuc | pAAV-UAS | 104840 |
P11 | IgK sp-HA-CD4-10aa linker-CIBN-NNES-NanoLuc | pAAV-CMV | 125229 |
P12 | gLuc sp-NanoLuc-HA-oβ2AR-TS-eLOV-TEVcs-Flag-Gal4 | pLX208-CMV | 125230 |
P13 | NanoLuc-15aa linker-βarrestin2-HA-GS linker-TEVp | pLX208-CMV | 125231 |
P14 | UAS-Citrine | pFPGW-UAS | 125232 |
P15 | gLuc sp-NanoLuc-15aa linker-ICAM-Nrxn3b-HA | pLX208-CMV | 125233 |
P16 | gLuc sp-GS linker-HiBit-Flag-oβ2AR-TS-NNES-eLOV-TEVcs-Flag-Gal4-V5 | pAAV-CMV | 125234 |
P17 | NanoLuc-15aa linker-βarrestin2-no HA-GS linker-TEVp | pAAV-CMV | 125235 |
P18 | β2AR-NNES-NanoLuc-eLOV-TEVcs-Flag-Gal4-V5 | pAAV-CMV | 125236 |