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PLos ONE 13(5), e0196946. β-Arrestin1 and 2 differentially regulate PACAP-induced PAC1 receptor signaling and trafficking. 2018

Shintani, Y., Hayata-Takano, A., Moriguchi, K., Nakazawa, T., Ago, Y., Kasai, A., Seiriki, K., Shintani, N. and Hashimoto, H.

Notes: The authors demonstrate that β-arrestin1 and β-arrestin2 differ in there interaction with PAC1R by using the Promega NanoBiT® technology. (5082)

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PLos ONE 13(4), e0195879. A novel nanoluciferase-based system to monitor Trypanosoma cruzi infection in mice by bioluminescence imaging. 2018

Silberstein, E., Serna, C., Fragoso, S.P., Nagarkatti, R., and Debrabant, A.

Notes: Infection progression remains poorly characterized in Chagas disease, a life-long infection caused by T. cruzi. To understand the development of the disease, a bioluminescent imaging system for live mice infected with NanoLuc-labeled transgenic T. cruzi was developed. Initial expression of NanoLuc luciferase was determined in TcCOL cells and cell lysates using the Nano-Glo Live Cell assay and the Nano-Glo Luciferase Assay System, respectively. Luminescence could be detected as early as 14 days post-infection and was focused in the abdomen. Further, luminescence remained detectable for up to 222 days post infection, while parasite number in blood decreased after 21 days to the limit of detection.  (5110)

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Anal. Biochem. 555, 67–72. Antibody-free detection of cellular neddylation dynamics of Cullin1. 2018

Schwinn, M.K., Hoang, T., Yang, X., Zhao, X., Ma, J., Li, P., Wood, K.V., Mallender, W.D., Bembenek, M.E. and Yan, Z.H.

Notes: The post-transcriptional modification of neddylation helps regulate protein activity, stability and localization. NanoLuc® Binary Technology (NanoBiT) was used to monitor covalent neddylation of Cul1 in a cellular context. SmBiT-Nedd8 and Cul1-LgBiT were expressed in HEK293 cells and luminescence was monitored as a response to a Nedd8-Cul1 covalent protein modification. Further, neddylation was monitored in the presence of both inhibitors and activators, and a concentration- and time-dependent response in luminescence was observed. (5076)

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FEBS J. 285, 46–71. Dynamic control of RSK complexes by phosphoswitch-based regulation. 2018

Gógl, G., Biri-Kovács, B., Póti, Á.L., Vadászi, H., Szeder, B., Bodor, A., Schlosser, G., Ács, A., Turiák, L., Buday, L., Alexa, A., Nyitray, L. and Reményi, A.

Notes: The NanoBiT® PPI MCS Starter System was used to build assays to study the interaction dynamics of RSK1 with ERK2 and MAGl-1 in live cells upon EGF treatment. Various structure-based mutations were studied by transiently transfecting cells with constructions using FuGENE® HD Reagent and assayed with Nano-Glo® Live Cell Reagent. The authors were able to determine which mutations in RSK1 affected MAGl-1 dissociation and determined that it was dependent on the presence of the PDZ-binding motif. (4969)

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Biochem. Pharmacol. 148, 298–307. Molecular dissection of the human A3 adenosine receptor coupling with β-arrestin2. 2018

Storme, J., Cannaert, A., Van Craenenbroeck, K. and Stove, C.P.

Notes: The NanoBiT Protein-Protein Interaction System was utilized to investigate the interaction of human A3 adenosine receptor, a G protein-coupled receptor, and β-arrestin2. Specifically, the importance of specific C-terminal phosphorylation sites for A3AR and β-arrestin2 coupling was determined using protein truncations. To determine the optimal fusion tags for this assay, multiple constructs were tested in the presence of an A3AR selective agonist. Unlike previous studies in rat models, C-terminal human A3AR truncations displayed no major impact on β-arrestin2 coupling. (5077)

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Cancer Immunol. Res. 6(8), 921–929. PD-L1 Binds to B7-1 Only In Cis on the Same Cell Surface. 2018

Chaudhri, A., Xiao, Y., Klee, A.N., Wang, X., Zhu, B. and Freeman, G.J.

Notes: NanoBiT Protein-Protein Interaction System was used to determine an interaction between Programmed death ligand 1 (PD-L1) and B7-1. PD-L1 is an encouraging target for cancer immunotherapy due to its involvement in mediating tumor immune evasion. Previous studies identified an interaction between PD-L1 and B7-1, however here the NanoBiT system was used to show binding between PD-L1 and B7-1 was specific when both are co-expressed on the same cell surface. Interestingly, similar co-expression of PD-L1 and B7-1 was found on tumor-infiltrating myeloid cells. Further, targeted PD-L1 mutants showed a significant decrease in B7-1 binding. (5072)

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J. Biol. Chem. 293(23), 8750–8760. Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly. 2018

O'Neill, S., Mathis, M., Kovačič, L., Zhang, S., Reinhardt, J., Scholz, D., Schopfer, U., Bouhelal, R. and Knaus, U.G.

Notes: The assembly of the NOX4- p22phox integral membrane complex, involved in electron transfer and reactive oxygen species generation, was investigated using the NanoBiT Protein-Protein Interaction System. A total of 31 Small BiT/Large BiT NOX4 or p22phox fusions were tested, and the most active pairs were further analyzed for catalytic activity. Residues involved in complex formation and catalysis were determined using mutational analysis and tested for luciferase activity. Together, the authors identified an approach for determining membrane protein assembly that can be used in the future as a drug discovery tool. (5074)

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Sci. Rep. 8(1). TDP-43 self-interaction is modulated by redox-active compounds Auranofin, Chelerythrine and Riluzole. 2018

Oberstadt, M., Stieler, J., Simpong, D.L., Römuß, U., Urban, N., Schaefer, M., Arendt, T. and Holzer, M.

Notes: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by formation of TDP-43 protein aggregates. The NanoBiT Protein-Protein Interaction System was used to measure self-interaction of TDP-43. Multiple tag locations were tested, and fusion of the Large BiT and Small BiT tags on the N-terminus resulted in the highest signal. Drug screening for potential ALS therapeutics was performed by monitoring for the disruption of the TDP-43 aggregates and loss of luciferase activity using the NanoBiT system. Drug screening resulted in the identification of Auranofin as a potential therapeutic, which showed redistribution of insoluble aggregates to PBS soluble protein. (5073)

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Anal. Chem. 89, 9527–36. Activity-based detection of consumption of synthetic cannabinoids in authentic urine samples using a stable cannabinoid reporter system. 2017

Cannaert, A., Franz, F., Auwärter, V. and Stove, C.P.

Notes: The NanoBiT® system was used to develop CB1 and CB2 receptor activation bioassays based on β-arrestin recruitment, and stable cell lines were generated for these assays. The cell lines were used to screen for synthetic cannabinoids in urine samples and assayed using the Nano-Glo® Live Cell Reagent. (4966)

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J. Med. Chem. 60, 4869–81. Design and discovery of N-(2-Methyl-5'-morpholino-6'-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A potent, selective, and efficacious RAF inhibitor targeting RAS mutant cancers. 2017

Nishiguchi, G.A., Rico, A., Tanner, H., Aversa, R.J., Taft, B.R., Subramanian, S., Setti, L., Burger, M.T., Wan, L., Tamez, V., Smith, A., Lou, Y., Barsanti, P.A., Appleton, B.A., Mamo, M., Tandeske, L., Dix, I., Tellew, J.E., Huang, S., Mathews Griner, L.A., Cooke, V.G., Van Abbema, A., Merritt, H., Ma, S., Gampa, K., Feng, F., Yuan, J., Wang, Y., Haling, J.R., Vaziri, S., Hekmat-Nejad, M., Jansen, J.M., Polyakov, V., Zang, R., Sethuraman, V., Amiri, P., Singh, M., Lees, E., Shao, W., Stuart, D.D., Dillon, M.P. and Ramurthy, S.

Notes: The major goal of this study was to identify selective RAF inhibitors that would suppress the RAF-MEK-ERKK pathway. A NanoBiT® cRAF:bRAF interaction assay was used to study the potency of identified compounds to stabilize this dimer using dose-response assays in transfected HCT116 cells (4967)

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eLife 6, e26857. Estrogen receptor coregulator binding modulators (ERXs) effectively target estrogen receptor positive human breast cancers. 2017

Raj, G.V., Sareddy, G.R., Ma, S., Lee, T.K., Viswanadhapalli, S., Li, R., Liu, X., Murakami, S., Chen, C.C., Lee, W.R., Mann, M., Krishnan, S.R., Manandhar, B., Gonugunta, V.K., Strand, D., Tekmal, R.R., Ahn, J.M. and Vadlamudi, R,K.

Notes: The authors are studying synthetic peptides (peptidomimetics) and their ability to modulate estrogen receptor signaling. The authors developed NanoBiT® PPI assays to study estrogen receptor and androgen receptor dimerization. Using these assays, they were able to demonstrate compound ERX-11 blocks estrogen receptor signaling and can specifically disrupt ER dimerization, but does not affect AR dimerization. The authors also developed a NanoBiT® assay to study the interaction of ER with the coregulator PELP1 and found this interaction could also be decreased with exposure to ERX-11. (4968)

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Nat. Biotechnol. 34, 760-767. A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. 2016

Chu, J., Oh, Y., Sens, A., Ataie, N., Dana, H., Macklin, J.J., Laviv, T., Welf, E.S., Dean, K.M., Zhang, F., Kim, B.B., Tang, C.T., Hu, M., Baird, M.A., Davidson, M.W., Kay, M.A., Fiolka, R., Yasuda, R., Kim, D.S., Ng, H.L. and Lin, M.Z.

Notes: The authors characterize a novel orange-red fluorescent protein (CyOFP1), which serves as an efficient acceptor for resonance energy transfer from NanoLuc® luciferase. An optimized fusion protein of CyOFP1 and NanoLuc® (Antares) was demonstrated to function as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. During fluorometry, 50,000 cells in 10 μL Live Cell Imaging Solution were loaded into glass capillary tubes and gently pelleted by centrifugation for 15 s at 500g. 10 μL of Live Cell Imaging Solution with 20 μM D-luciferin, coelenterazine, ViviRen, or furimazine was then added. For BRET6 and Antares-expressing mice, tail vein injections were performed with 333 nmol (123 μg) of furimazine in 120 μL of 8% glycerol, 12% ethanol, 10% hydroxypropyl-β-cyclodextrin, and 35% polyethylene glycol 400 in water. (4760)

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Anal. Chem. 88, 11476–85. Detection and activity profiling of synthetic cannabinoids and their metabolites with a newly developed bioassay. 2016

Cannaert, A., Storme, J., Franz, F., Auwärter, V. and Stove, C.P.

Notes: The authors used NanoLuc® Binary Technology (NanoBiT®) to build β-arrestin recruitment assays for two G-protein coupled receptors (CB1 and CB2) to create live-cell cannabinoid reporter assays. The assays were used to compare activity of synthetic cannabinoids and their metabolites and also to screen urine for CB receptor activity using the Nano-Glo® Live Cell Assay System. (4965)

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ACS Chemical Biology 11, 400–408. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. 2016

Dixon, A.S., Schwinn, M.K., Hall, M.P., Zimmerman, K., Otto, P., Lubben, T.H., Butler, B.L., Binkowski, B.F., Machleidt, T., Kirkland, T.A., Wood, M.G., Eggers, C.T., Encell, L.P., Wood, K.V.

Notes: These authors designed a complementation reporter for detecting protein interactions under physiological conditions in mammalian cells. The engineered reporter (NanoBiT) is based on NanoLuc luciferase and is composed of two small subunits (18kDa and 1.3kDa), which associate weakly. The formation of a luminescent signal is dependent on the interaction characteristics of the proteins onto which the subunits are attached. The paper demonstrates utility of the NanoBiT assay for detecting protein interactions in live cells using  FRB/FKBP as an example, shows that response dynamics are rapid and reversible, and also illustrates use of the system to measure the pharmacology of kinase inhibitors that induce interaction between BRAF and CRAF. (4585)

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