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The NanoBiT-based β-arrestin recruitment assays. ( A ) Activation effect of recombinant Lo-GPR15LG towards <t>Lo-GPR15.</t> Inner panel, dose-response curve. ( B ) Activation effect of the C-terminally truncated Lo-[desC4]GPR15LG towards Lo-GPR15. ( C ) Activation effect of Hs-GPR15LG towards Lo-GPR15. ( D ) Activation effect of Lo-GPR15LG towards Hs-GPR15. In these β-arrestin recruitment assays, NanoLuc substrate and indicated peptide were sequentially added to living HEK293T cells coexpressing SmBiT-ARRB2 and indicated C-terminally LgBiT-fused GPR15 receptor, and bioluminescence was measured on an iD3 plate reader. Blue arrows indicate addition of NanoLuc substrate and red arrows indicate addition of peptide.
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The NanoBiT-based β-arrestin recruitment assays. ( A ) Activation effect of recombinant Lo-GPR15LG towards Lo-GPR15. Inner panel, dose-response curve. ( B ) Activation effect of the C-terminally truncated Lo-[desC4]GPR15LG towards Lo-GPR15. ( C ) Activation effect of Hs-GPR15LG towards Lo-GPR15. ( D ) Activation effect of Lo-GPR15LG towards Hs-GPR15. In these β-arrestin recruitment assays, NanoLuc substrate and indicated peptide were sequentially added to living HEK293T cells coexpressing SmBiT-ARRB2 and indicated C-terminally LgBiT-fused GPR15 receptor, and bioluminescence was measured on an iD3 plate reader. Blue arrows indicate addition of NanoLuc substrate and red arrows indicate addition of peptide.

Journal: bioRxiv

Article Title: Origin of the GPR15LG–GPR15 signaling axis in ancient fish ancestors

doi: 10.64898/2026.06.02.729464

Figure Lengend Snippet: The NanoBiT-based β-arrestin recruitment assays. ( A ) Activation effect of recombinant Lo-GPR15LG towards Lo-GPR15. Inner panel, dose-response curve. ( B ) Activation effect of the C-terminally truncated Lo-[desC4]GPR15LG towards Lo-GPR15. ( C ) Activation effect of Hs-GPR15LG towards Lo-GPR15. ( D ) Activation effect of Lo-GPR15LG towards Hs-GPR15. In these β-arrestin recruitment assays, NanoLuc substrate and indicated peptide were sequentially added to living HEK293T cells coexpressing SmBiT-ARRB2 and indicated C-terminally LgBiT-fused GPR15 receptor, and bioluminescence was measured on an iD3 plate reader. Blue arrows indicate addition of NanoLuc substrate and red arrows indicate addition of peptide.

Article Snippet: G protein-coupled receptor 15 (GPR15) is predominantly expressed in specific immune cell populations, including T cells, B cells, and plasma cells, according to data from the Human Protein Atlas ( https://www.proteinatlas.org ).

Techniques: Activation Assay, Recombinant

The NanoBiT-based ligand–receptor binding assays. ( A ) Activity of the recombinant SmBiT-Lo-GPR15LG towards Lo-GPR15 measured via the NanoBiT-based β-arrestin recruitment assay. Inner panel , dose-response curve. NanoLuc substrate and different concentrations of SmBiT-Lo-GPR15LG were sequentially added to living HEK293T cells coexpressing Lo-GPR15-LgBiT and SmBiT-ARRB2, and bioluminescence was measured on a plate reader. Blue arrow indicates addition of NanoLuc substrate, and red arrow indicates addition of peptide. ( B , C ) Saturation binding of SmBiT-Lo-GPR15LG with sLgBiT-Lo-GPR15 without (B) or with (C) coexpression of human TPST1 and TPST2. The measured bioluminescence data are expressed as mean ± SD ( n = 3) and plotted using the SigmaPlot10.0 software. Total binding data (green circles) were fitted with the function of Y = B max X/(K d +X), non-specific binding data (red triangles) were fitted with linear curves. ( D ) The NanoBiT-based competition binding assays. The relative bioluminescence data are expressed as mean ± SD ( n = 3) and fitted with sigmoidal curves using the SigmaPlot10.0 software.

Journal: bioRxiv

Article Title: Origin of the GPR15LG–GPR15 signaling axis in ancient fish ancestors

doi: 10.64898/2026.06.02.729464

Figure Lengend Snippet: The NanoBiT-based ligand–receptor binding assays. ( A ) Activity of the recombinant SmBiT-Lo-GPR15LG towards Lo-GPR15 measured via the NanoBiT-based β-arrestin recruitment assay. Inner panel , dose-response curve. NanoLuc substrate and different concentrations of SmBiT-Lo-GPR15LG were sequentially added to living HEK293T cells coexpressing Lo-GPR15-LgBiT and SmBiT-ARRB2, and bioluminescence was measured on a plate reader. Blue arrow indicates addition of NanoLuc substrate, and red arrow indicates addition of peptide. ( B , C ) Saturation binding of SmBiT-Lo-GPR15LG with sLgBiT-Lo-GPR15 without (B) or with (C) coexpression of human TPST1 and TPST2. The measured bioluminescence data are expressed as mean ± SD ( n = 3) and plotted using the SigmaPlot10.0 software. Total binding data (green circles) were fitted with the function of Y = B max X/(K d +X), non-specific binding data (red triangles) were fitted with linear curves. ( D ) The NanoBiT-based competition binding assays. The relative bioluminescence data are expressed as mean ± SD ( n = 3) and fitted with sigmoidal curves using the SigmaPlot10.0 software.

Article Snippet: G protein-coupled receptor 15 (GPR15) is predominantly expressed in specific immune cell populations, including T cells, B cells, and plasma cells, according to data from the Human Protein Atlas ( https://www.proteinatlas.org ).

Techniques: Binding Assay, Activity Assay, Recombinant, Software