Journal: Cell

Article Title: Human Gain-of-Function MC4R Variants Show Signaling Bias and Protect against Obesity

doi: 10.1016/j.cell.2019.03.044

Figure Lengend Snippet:

Article Snippet: GloSensor cAMP biosensor - pGloSensor 20F plasmid , Promega , Cat#E1171.

Techniques: Virus, Recombinant, Plasmid Preparation, Software

Journal: Pharmacology Research & Perspectives

Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

doi: 10.1002/prp2.1223

Figure Lengend Snippet: NanoBRET binding curves for PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. (A) Cells were incubated with increasing concentrations of PSB603‐BY630 in the absence or presence of 1 μM MRS‐1706. (B) Specific‐binding of PSB603‐BY630 to NLuc‐A 2B Rs. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). The mean K D value obtained in five separate experiments was 18.32 ± 1.65 nM.

Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

Techniques: Binding Assay, Expressing, Incubation

Journal: Pharmacology Research & Perspectives

Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

doi: 10.1002/prp2.1223

Figure Lengend Snippet: NanoBRET imaging of PSB603‐BY630 binding to HEK293G cells expressing NLuc‐A 2B R. Cells were incubated with 100 nM PSB603‐BY630 in the absence (A, B) or presence (C, D) of 10 μM MRS‐1706 before addition of furimazine (1:800 dilution) and imaging. Filtered bioluminescence was captured using a 438/24 bandpass filter (cyan A & C). BRET was captured using a 650/50 nm bandpass filter (magenta B & D). Images are representative of those obtained in five independent experiments. Scale bar represents 100 μm.

Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

Techniques: Imaging, Binding Assay, Expressing, Incubation

Journal: Pharmacology Research & Perspectives

Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

doi: 10.1002/prp2.1223

Figure Lengend Snippet: NanoBRET competition binding in HEK293G cells exogenously expressing NLuc‐A 2B R. Cells were incubated with 50 nM PSB603‐BY630 in the absence or presence of competing ligands Data are mean ± S.E.M. from five independent experiments. The open and closed bars show the BRET ratio obtained in the absence and presence of 50 nM PSB603‐BY630 respectively.

Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

Techniques: Binding Assay, Expressing, Incubation

Journal: Pharmacology Research & Perspectives

Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

doi: 10.1002/prp2.1223

Figure Lengend Snippet: Ligand‐binding kinetics of 200 nM PSB603‐BY630 in HEK293G cells exogenously expressing NLuc‐A 2B R. BRET ratios for the total binding of 200 nM PSB603‐BY630 were obtained every 30 sec. In parallel, data were also collected in the absence of fluorescent ligand for each time point and these data were subtracted from the total binding to obtain baseline‐corrected values for total binding at each time point. Sixty minutes after addition of 200 nM PSB603‐BY630, 10 μM MRS‐1706 was added to initial dissociation of the fluorescent ligand. Values show mean ± S.E.M of quadruplicate determinations in a single representative experiment. Similar data were obtained in four other experiments. The data points for the dissociation phase of the experiment were then fitted to a single exponential function to determine the the dissociation rate constant ( k off ) in min −1 as described under Methods. In this representative experiment the calculated K off value was 0.056 min −1 . The mean K off value obtained in the five repeat experiments was 0.065 ± 0.003 min ‐1 .

Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

Techniques: Ligand Binding Assay, Expressing, Binding Assay

Journal: Pharmacology Research & Perspectives

Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

doi: 10.1002/prp2.1223

Figure Lengend Snippet: NanoBRET binding curves for PSB603‐BY630 and receptor‐selective fluorescent ligands binding to NLuc‐tagged A 1 , A 2A or A 3 adenosine receptors. (A, B) Total and non‐specific binding of (A) PSB603‐BY630 or (B) EC‐005 to transiently transfected NLuc‐A 2A R obtained in the absence and presence of 1 μM of the A 2A R‐selective antagonist SCH58261. Data are mean ± S.E.M obtained in five independent experiments (each conducted in duplicate). (C, D) Total and non‐specific binding of (C) PSB603‐BY630 or (D) EC‐069 to NLuc‐A 1 R in a stable HEK293T cell line obtained in the absence and presence of 1 μM of the A 1 R‐selective antagonist SLV320. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate). (E, F) Total and non‐specific binding of (E) PSB603‐BY630 or (F) AV‐039 to NLuc‐A 3 R in a stable HEK293G cell line obtained in the absence and presence of 1 μM of the A 3 R‐selective antagonist MRS‐1220. Data are mean ± S.E.M obtained in five independent experiments (each conducted in triplicate).

Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

Techniques: Binding Assay, Transfection

Journal: Pharmacology Research & Perspectives

Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

doi: 10.1002/prp2.1223

Figure Lengend Snippet: Effect of PSB603‐BY630 on Glosensor cAMP concentration‐response curves to the A 2B ‐selective agonist BAY 60–6583 in (A) HEK293G cells endogenously expressing A 2B R or (B) HEK293G cells overexpressing NLuc‐A 2B R. Concentration response curves were obtained in the absence and presence of 20 nM or 200 nM PSB603‐BY630. Values are mean ± S.E.M. of five separate experiments carried out in triplicate. Data represent peak luminescence response and are expressed as a percentage of the peak luminescence response to 3 μM BAY 60–6583 (in a) or 0.3 μM BAY 60–6583 (in b) obtained in the absence of antagonist in each individual experiment.

Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

Techniques: Concentration Assay, Expressing

Journal: Pharmacology Research & Perspectives

Article Title: A novel and selective fluorescent ligand for the study of adenosine A 2B receptors

doi: 10.1002/prp2.1223

Figure Lengend Snippet: Log EC 50 and E MAX values obtained in HEK293G cells endogenously expressing A 2B R or HEK293G cells expressing recombinant human A 2B R for BAY 60–6593 obtained in the absence and presence of increasing concentrations of PSB603BY630.

Article Snippet: Dimethyl Sulfoxide (DMSO) (Cat#D5879), lipopolysaccharide (LPS) (Cat# L2654) and Bovine Serum Albumin (BSA) (Cat# A7030) were purchased from Sigma‐Aldrich (Gillingham, UK).The cAMP GloSensor™ Human Embryonic Kidney 293 (HEK293G) cell line, the Nano‐Glo® Luciferase Assay System and GloSensor™ cAMP reagent were purchased from Promega Corporation (Madison, WI, USA).

Techniques: Expressing, Recombinant

Journal: FASEB BioAdvances

Article Title: Fusion of the β 2 ‐adrenergic receptor with either Gαs or βarrestin‐2 produces constitutive signaling by each pathway and induces gain‐of‐function in BEAS‐2B cells

doi: 10.1096/fba.2022-00038

Figure Lengend Snippet: Gαs activity and βarr2 recruitment by the fusion proteins β 2 AR‐Gαs and β 2 AR‐βarr2 in HEK 293 cells. (A) Schematic representation of the Gαs activity ( upper panel ) using GloSensor technology. By increasing the concentrations of cAMP, the genetically engineered firefly luciferase undergoes a conformational change that induces luminescence. Here, the Gαs activity is measured by its capacity to produce cAMP after stimulation of the β 2 AR with isoproterenol (ISO) ( lower panel ). The wild‐type β 2 AR (blue) shows a time‐dependent increase in luminescence after stimulation with 10 μM ISO. Such activity is markedly diminished when the β 2 AR is fused to either Gαs (red) or βarr2 (green). (B) Schematic representation of the βarr2 recruitment using a structural complementation assay adapted to NanoBiT technology by molecular cloning ( upper panel ). β 2 AR‐LgBiT (blue) recruits SmBiT‐βarr2 after 10 μM isoproterenol (ISO) stimulation ( lower panel ). This recruitment is drastically blunted when the receptor is fused to either Gαs (red) or βarr2 (green) ( lower panel ). All experiments are expressed as the mean ± SEM from three independent experiments.

Article Snippet: HEK293 cells were plated at 35,000 cells per well in white bottom 96‐well plates, and 24 h thereafter, each well was transiently transfected with 50 ng of GloSensor biosensor and 50 ng of pcDNA3.1+‐β 2 AR, ‐β 2 AR‐βarr2, or ‐β 2 AR‐Gαs using ViaFectTM Transfection Reagent (Promega Cat. No. E4982).

Techniques: Activity Assay, Luciferase, Molecular Cloning