Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: a The cubic ternary complex model for a GPCR interacting with agonists (A), inverse agonists (I) and Gs alpha subunits (Gs). In this model, the receptor can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and Gs alpha subunits. The conformational equilibrium that exists between R and R* (red arrows) explains the concept of constitutive receptor activity whereby basal activity can be observed in the absence of agonists in cells overexpressing native or constitutively active mutant β 2 ARs, as a consequence of binding to Gs proteins. b The mechanical mechanism for loading the multi-well plate into the PheraStar plate reader. The 96-well plate is placed on the mechanical plate stage and then automatically taken into the plate reader with a simple motor-driven linear movement. This process has the potential to provide a linear mechanical stimulation of the cell monolayer as the plate enters the light-tight reader. The initial entry of the plate into the reader is normally sufficient to activate the receptor. In most experiments, an initial read was taken at time zero and then the plate was immediately removed for the addition of ligands before re-entering the reader. In some experiments, the plate was added slowly manually to gain an initial GloSensor TM reading of basal levels. Created with Biorender.com.

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Activity Assay, Mutagenesis, Binding Assay

Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: Time-course of GloSensor TM luminescence stimulated by increasing concentrations of a isoprenaline and b salbutamol in a clonal HEK293G cell line overexpressing a transfected human TS-SNAP-β 2 AR. An initial luminescence read was made at time zero. The plate was then immediately removed from the PheraStar, agonists or HBSS added and the plate was then returned to the PheraStar and measurements continued every 1 min for 60 min. Values are mean ± SEM of 5 independent experiments. In each individual experiment, triplicate determinations were made. c Concentration–response curves of peak luminescence responses obtained for formoterol, isoprenaline, salmeterol and salbutamol in HEK293G cells overexpressing recombinant TS-SNAP-β 2 AR. Data are expressed as a percentage of the response to 1 nM isoprenaline (after normalisation of HBSS control response to zero) obtained in each individual experiment and represent mean ± SEM from five independent experiments ( n = 5). d A comparison of the time-course of GloSensor TM basal responses to HBSS addition in HEK293G cells overexpressing recombinant TS-SNAP-β 2 AR or wild-type (WT) HEK293G cells expressing endogenous β 2 ARs at very low levels. Values are mean ± SEM from five independent experiments. e , f Data from d showing the response in each cell line at t = 0 min prior to the addition of HBSS ( e ) and at the peak of the response to HBSS ( f ). ** p = 0.0079 (Mann–Whitney U test). At t = 0, there was no significant difference between the two cell lines ( p = 0.15; Mann–Whitney U test). Outlier analysis (both ROUT and Grubs method) confirmed that there were no significant outliers in the data sets. It should be noted that in these experiments, the zero time points were taken following initial plate loading and the plate was then immediately removed for the addition of an agonist or HBSS, and the plate was then reinserted into the plate reader.

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Transfection, Concentration Assay, Recombinant, Control, Comparison, Expressing, MANN-WHITNEY

Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: Agonist E max , and log EC 50 determined for isoprenaline, formoterol, salbutamol and salmeterol from concentration–response curves obtained by cAMP GloSensor TM in HEK293G cells expressing endogenous β 2 ARs or HEK293G cells overexpressing recombinant TS-SNAP-β 2 AR

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Concentration Assay, Expressing, Recombinant

Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: Impact of mechanical stimulation on basal GloSensor TM time-course responses in a clonal HEK293G cell line overexpressing recombinant TS-SNAP-β 2 AR ( a , b ) or HEK293G cells endogenously expressing β 2 ARs ( c ). In all experiments, an initial luminescence read was made at time zero after initial plate entry into the PheraStar and before any additions. The plate was then immediately removed, HBSS or ICI-118551 added, and the plate was then returned to the PheraStar. Measurements were then made at 1 min and every min for 60 min in total. In a and c HBSS or ICI-118551 (1 μM) were added, and measurements of luminescence continued every minute from time = 1 min. a , c At 15, 30 and 45 min, the motorised stage of the PheraStar removed the plate from the instrument and then immediately returned it to the plate reader for further measurements every minute. In b no additions were made before measurements were made, although the motorised stage removed the plate and returned it to the PheraStar immediately after the time = 0 initial read to be consistent with the experiments in ( a ) and ( c ). At 30 min, the motorised stage of the PheraStar removed the plate from the instrument, HBSS or 1 μM ICI-118551 was added and the plate was then immediately returned to the plate reader for further measurements every min. In a – c values are mean ± SEM from five independent experiments. In each individual experiment, triplicate determinations were made.

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Recombinant, Expressing

Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: a , b Time-course of the basal GloSensor TM responses in HEK293G cells expressing HiBiT-D113A-β 2 AR, HEK293G cells expressing HiBiT-β 2 AR wild-type or native HEK293G cells with endogenous-β 2 ARs obtained in the absence ( a ) and presence ( b ) of 1 μM ICI-118551. In both ( a ) and ( b ), the plate was placed in the PheraStar at t = −15 min, the plate was then removed at time zero and HBSS or ICI-118551 (1 μM) was added, and the plate immediately returned to the PheraStar. Values are mean ± SEM from seven independent experiments. c Comparison of maximal basal peak responses obtained in HEK293G cells over-expressing HiBiT-D113A-β 2 AR, HEK293G cells over-expressing HiBiT-β 2 AR wild-type or in native HEK293G cells with endogenous expression of β 2 ARs. Values are mean ± SEM from seven independent experiments. * p < 0.05 or ** p < 0.01 (one-way ANOVA with Holm-Sidak multiple comparison test). HEK293G-HiBiT-D113A-β 2 AR versus HEK293G-HiBiT-D113A-β 2 AR in the presence of 1 μM ICI-118551 (not significant, p = 0.32); HEK293G-HiBiT-D113A-β 2 AR plus ICI-118551 versus endogenous HEK293G cells plus or minus ICI-118551 (both p = 0.035); HEK293G-HiBiT-D113A-β 2 AR plus 1 μM ICI-118551 versus wild-type β 2 AR ( p = 0.009). d Cell surface expression of HiBiT-D113A-β 2 AR in HEK293G cells. Receptor expression was monitored as reconstituted nanoluciferase luminescence following the addition of 0.2% purified LgBiT and 0.2% furimazine. Values are mean ± SEM from six independent experiments. * p < 0.05 or ** p < 0.01 (ANOVA with Tukey’s multiple comparison test for matched data). P = 0.045 and 0.005 for HiBiT-β 2 AR and HiBiT-D113A-β 2 AR, respectively relative to untransfected HEK293G cells. e Specific binding of ICI-118,551-βAla-βAla-BODIPY-X-630/650 (fluorescent ICI-118551) to HEK293G cells expressing wild-type HiBiT-β 2 AR or HiBiT-D113A-β 2 AR. Total and non-specific binding was determined following the re-complementation of full-length nanoluciferase with the addition of 0.2% purified LgBiT. Non-specific binding was determined in the presence of 50 μM ICI-118551. Specific binding was determined by subtraction of non-specific binding from the total binding at each concentration of fluorescent ICI-118551. Values are mean ± SEM from five independent experiments. f Specific binding determined with 100 nM fluorescent ICI-118551 in HEK293G cells expressing wild-type HiBiT-β 2 AR or HiBiT-D113A-β 2 AR. Data taken from ( e ) showing mean ± SEM and the individual means obtained in five separate experiments. **** p < 0.0001 (paired t -test).

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Expressing, Comparison, Purification, Binding Assay, Concentration Assay

Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: Cells were transiently transfected with the wild-type (WT) β 2 AR, a double mutant (N6A and N15A) β 2 AR or a triple mutant ((N6A, N15A, N178A) β 2 AR or a pcDNA3.1 control. Each β 2 AR construct contained an N-terminal HiBiT sequence. a Comparison of the cell surface expression of the three β 2 AR constructs. Receptor expression was monitored as reconstituted nanoluciferase luminescence following the addition of 0.2% purified LgBiT and 0.25% furimazine. Values are mean ± SEM from six independent experiments. There was no significant difference between the expression levels (one-way ANOVA with Tukey’s multiple comparison tests; p = 0.923, p = 0.252 and P = 0.427 for WT versus double mutant, WT versus triple mutant, and double mutant versus triple mutant, respectively). b Comparison of maximal peak responses to 1 μM isoprenaline produced by the three β 2 AR constructs. Values are mean ± SEM from six independent experiments. There was no significant difference between the maximal responses (one-way ANOVA with Tukey’s multiple comparisons test; p = 0.844, p = 0.946 and P = 0.663 for WT versus double mutant, WT versus triple mutant and double mutant versus triple mutant, respectively). c Time-course and d peak basal GloSensor TM responses in the presence and absence of 1 μM ICI-118551. An initial luminescence read was made at time zero. The plate was then immediately removed, HBSS or ICI-118551 added and then the plate was returned to the PheraStar. Measurements were then made at 1 min and every min for 60 min in total. Values are mean ± SEM from six independent experiments. **** p < 0.0001, ** p = 0.005; * p = 0.023 (two-way ANOVA with Tukey’s multiple comparison test). 1 μM ICI-118551 had no significant effect on the basal response to the triple mutant ( p = 0.595). e Time-course and f peak basal GloSensor TM responses in the presence and absence of 1 μM ICI-118551 or 1 μM isoprenaline f in HEK293 cells transfected with the triple β 2 AR mutant or pcDNA3.1. Values are mean ± SEM from six independent experiments. **** p < 0.0001 (two-way ANOVA with Sidak’s multiple comparisons test). The peak responses to isoprenaline were not significantly different ( p = 0.380). The peak responses to HBSS were also not significantly different ( p > 0.99). In both the triple mutant ( p > 0.77) and the pcDNA3.1 control cells ( p > 0.98), the inhibition by 1 μM ICI-118551 did not reach significance.

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Transfection, Mutagenesis, Control, Construct, Sequencing, Comparison, Expressing, Purification, Produced, Inhibition

Journal: bioRxiv

Article Title: Antagonistic interactions between odorants alter human odor perception

doi: 10.1101/2022.08.02.502184

Figure Lengend Snippet: A) Dose-response curves of OR2T11 against increasing concentrations of ionones and damascones, with the vapor concentration of CH 3 SH held constant at 7 ppm. IC50 value was calculated using Graph pad Prism software. B) Antagonistic effect of damascone & ionone analogs. 41 ppm H 2 S stimulation on human OR2T1, OR2T6 and OR2T11 were masked by 100 µM fragrance compounds. Multiple comparisons were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (*** p <0.001). C) Inhibitor odorants did not cause adverse effects on the assay system. OR2T11 and mouse Or2aj6 (Olfr171) (damascones responding receptor) expressing cells were stimulated by 100µM odorants and Glosensor buffer without any odorant as a negative control. Error bars indicate s.e.m (n=3) Multiple comparisons were performed using one-way ANOVA followed by Dunnett’s test (*** p <0.001).

Article Snippet: 18-24 hours later, the medium was replaced with 25 μL of HBSS (Gibco) containing 10 mM HEPES and 1 mM Glucose, followed by 25 μL of the HBSS containing GloSensor cAMP Reagent (Promega).

Techniques: Concentration Assay, Software, Expressing, Negative Control

Journal: Toxins

Article Title: Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin

doi: 10.3390/toxins13070480

Figure Lengend Snippet: Human α-defensin-1 and -5 inhibit PT-mediated effects on cAMP signaling. PT (100 ng/mL) was pre-incubated with 12 µM α-defensin-1, α-defensin-5 or ß-defensin-1, or with the respective amount of solvent (H 2 O) for 15 min at room temperature and then added to iGIST sensor cells for 5 h at 37°C. For further control, cells were treated only with the solvent of PT. Then, inducing medium containing the luciferase substrate for the luminescent biosensor for cAMP was added. After 15 minutes of baseline measurement, cells were spiked with forskolin to activate adenylate cyclase and octreotide acetate to activate Gαi-coupled SSTR2 GPCR. Luminescence was recorded for further 60 minutes. ( a ) cAMP kinetic curves from one representative experiment are shown as mean ± SD (n = 3 from one experiment), con = cells treated only with forskolin (FSK) plus octreotide (Oct). ( b ) Bar graphs show baseline-subtracted area under the curve (AUC) from at least three independent experiments. Values are given as percent of PT-only-treated samples, mean ± SEM (n = at least nine from at least three independent experiments). Values for control samples are identical in both graphs. Results are shown in two separate graphs for better clarity. Significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to samples treated with PT only (left graph) or untreated controls (right graph) (** p ≤ 0.01,**** p ≤ 0.0001, ns not significant). ( c ) For control, iGIST sensor cells were treated only with FSK or with PT plus FSK in the absence of Oct to measure the maximal cAMP response in this assay. Values for control samples and samples treated with PT plus FSK and Oct are identical to values in ( a ).

Article Snippet: HEK293 cells (HEK-Gs/SSTR2_HA), ectopically expressing Gαi-coupled somatostatin receptor 2 (SSTR2) GPCR as well as a luminescence cAMP probe (GloSensor-22F, Promega, Mannheim, Germany) [ ], were cultivated in DMEM/F12 (containing glutamine and sodium pyruvate) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen) and penicillin–streptomycin (1:100) (Thermo Fisher Scientific).

Techniques: Incubation, Solvent, Control, Luciferase, Comparison

Journal: Nature Communications

Article Title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

doi: 10.1038/s41467-022-32390-1

Figure Lengend Snippet: a Schematic of DREADD-β2AR and corresponding protein sequence encoding for signal peptide (black), VSV-G epitope (magenta), hM3Dq ligand binding domains (blue), and β2AR signaling domains (green). Black arrow: start of the mature GPCR after post-translational cleavage of the signal peptide. b Orthogonal view of DREADD-β2AR-transfected HEK cells immunostained for the N-terminal VSV-G tag under non-permeabilizing conditions. Magenta: VSV-G tag. Blue: nuclear staining with Hoechst. CMV, human cytomegalovirus promoter. c Schematic of signaling pathways for functional validation of the DREADD-based chimeras. The heterotrimeric G protein consists of an α- and βγ-subunit. Below: hM3Dq is a Gα q -coupled receptor, whereas non-chimeric β2AR recruits G proteins with a Gα s subunit. DREADD-β2AR contains the β2AR signaling domains to recruit Gα s . The DREADDs rM3Ds couples to Gα s and hM4Di is associated with Gα i . d Schematic of Gα s -coupled GPCR inducing cAMP synthesis after ligand stimulation through adenylyl cyclase (AC) activation. e – g Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-β2AR ( e ); non-chimeric β2AR ( f ), hM3Dq, rM3Ds or hM4Di ( g ); or empty vector ( e – f ). Baseline measurements of 30 min (first 15 min not shown) followed by ligand application (gray arrow for onset) of either CNO or levalbuterol. Measure of center: Mean fold change compared to baseline mean (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four (DREADD-β2AR: CNO 0.1–10 µM), seven (Empty vector: CNO), four (Non-chimeric β2AR: Levalbuterol; Empty vector: Levalbuterol; hM3Dq: CNO; hM4Di: CNO), or three (rM3Ds: CNO) experimental repetitions. Source data are provided as a Source Data file. h Schematic of Gα q -coupled GPCR engaging in the mitogen-activated protein kinase (MAPK) pathway which induces transcription of a firefly luciferase reporter from a serum responsive element (SRE). i Endpoint measurement of SRE-dependent luciferase activity in HEK cells transfected with hM3Dq (blue), DREADD-β2AR (magenta), non-chimeric β2AR (green), rM3Ds (red), hM4Di (cyan), or empty vector (gray). Ligand stimulation either with 10 µM CNO (left) or 0.001 µM levalbuterol (right). Dashed line: level of vehicle control. Error bars: standard error of the mean. Two-sided one-sample T-test for comparing to a mean of 1 representing the vehicle control: *** p < 0.001; ** p < 0.01; n.s. p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.004 (hM3Dq: CNO); p = 0.03 (rM3Ds: CNO); p = 0.001 (hM4Di: CNO); p < 0.002 (DREADD-β2AR: CNO); p = 0.09 (Empty vector: CNO); p < 0.001 (Non-chimeric β2AR: Levalbuterol); p = 0.01 (Empty vector: Levalbuterol). N = three experimental repetitions. Source data are provided as a Source Data file.

Article Snippet: To measure Gα i -induced decreases in cAMP, we utilized a cAMP-dependent firefly luciferase suitable for Gα i signaling (GloSensor-22F; Promega; E2301) .

Techniques: Sequencing, Ligand Binding Assay, Transfection, Staining, Protein-Protein interactions, Functional Assay, Biomarker Discovery, Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

doi: 10.1038/s41467-022-32390-1

Figure Lengend Snippet: a Schematic of GPCR increasing baseline cAMP levels through constitutive activity. b Real-time measurement of cAMP-dependent luciferase activity during a 30 min baseline in HEK cells transfected with hM3Dq (blue), rM3Ds (red), hM4Di (cyan), DREADD-β2AR (magenta), or non-chimeric β2AR (green). Measure of center: Mean fold change compared to empty vector (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four experimental repetitions. Source data are provided as a Source Data file. c Graph shows average fold changes compared to empty vector control during the 30 min measurement in ( b ). Dashed line: level of empty vector control. Error bars: standard error of the mean. N = four experimental repetitions. Two-sided one-sample T -test for comparing to a mean of 1 representing the empty vector control: *** p < 0.001; ** p < 0.01; n.s. p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.81 (hM3Dq); p = 0.009 (rM3Ds); p = 0.48 (hM4Di); p = 0.01 (DREADD-β2AR); p = 0.009 (Non-chimeric β2AR). Source data are provided as a Source Data file. d Schematic of GPCR with constitutive activity impacting baseline MAPK signaling measured through an SRE reporter. e Endpoint measurement of SRE-dependent luciferase activity in transfected HEK cells. Dashed line: level of empty vector control. Error bars: standard error of the mean. N = seven (hM3Dq, DREADD-β2AR), four (rM3Ds, hM4Di), or nine (Non-chimeric β2AR) experimental repetitions. of four to nine repetitions. Two-sided one-sample T -test for comparing to a mean of 1 representing the empty vector control: *** p < 0.001; ** p < 0.01; n.s. p > 0.05. Exact p-values of individual T -tests without multiple testing correction: p < 0.001 (hM3Dq); p < 0.001 (rM3Ds); p = 0.30 (hM4Di); p < 0.001 (DREADD-β2AR); p < 0.001 (Non-chimeric β2AR). Source data are provided as a Source Data file.

Article Snippet: To measure Gα i -induced decreases in cAMP, we utilized a cAMP-dependent firefly luciferase suitable for Gα i signaling (GloSensor-22F; Promega; E2301) .

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

doi: 10.1038/s41467-022-32390-1

Figure Lengend Snippet: a , b Orthogonal view of HEK cells transfected with DREADD-GPR65 ( a ) or DREADD-GPR109A ( b ) immunostained for the VSV-G tag under non-permeabilizing conditions. Magenta: VSV-G tag. Blue: nuclear staining with Hoechst. CMV, human cytomegalovirus promoter. c Top: Schematic of Gα s -coupled GPCR inducing cAMP synthesis after ligand stimulation through adenylyl cyclase (AC) activation. Below: Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-GPR65 (magenta) or empty vector (gray). Baseline measurement of 30 min (first 15 min not shown) followed by CNO application (gray arrow for onset). Measure of center: Mean fold change compared to baseline mean (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four (DREADD-GPR65) or seven (Empty vector) experimental repetitions. Source data are provided as a Source Data file. d Endpoint measurement of serum responsive element (SRE)-dependent luciferase activity in HEK cells transfected with DREADD-GPR65 (magenta) or empty vector (gray). Ligand stimulation with CNO. Dashed line: level of the respective vehicle control. Error bars: standard error of the mean. Two-sided one-sample T -test for comparing to a mean of 1 representing the vehicle control: * p < 0.05; n.s . p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.009 (DREADD-GPR65); p = 0.09 (Empty vector). N = four (DREADD-GPR65) or three (Empty vector) experimental repetitions. Source data are provided as a Source Data file. e Top: Schematic of Gα i -coupled GPCR reducing cAMP levels after ligand stimulation through adenylyl cyclase (AC) inhibition. Forskolin induces cAMP synthesis through AC activation. Below: Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-GPR109A (magenta) or empty vector (gray). Baseline measurements followed by application of CNO or vehicle (gray arrow for onset) and forskolin (white arrow for onset). Measure of center: Mean fold change compared to vehicle (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = five experimental repetitions. Source data are provided as a Source Data file. f Schematic of competition assay between Gα i -coupled DREADD-GPR109A and Gα s -coupled A2B adenosine receptor (A2BAR). Simultaneous stimulation of Gα i through CNO and Gα s through NECA prevents cAMP-responsive element (CRE)-mediated luciferase reporter activity. g Endpoint measurement of CRE-dependent luciferase activity in HEK cells transfected with DREADD-GPR109A (magenta) or empty vector (gray). Simultaneous stimulation with CNO and 5 µM NECA. Dashed line: level of the respective vehicle control. Error bars: standard error of the mean. Two-sided one-sample T -test for comparing to a mean of 1 representing the vehicle control: * p < 0.05; n.s. p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.03 (DREADD-GPR109A); p = 0.34 (Empty vector). N = three (DREADD-GPR109A) or four (Empty vector) experimental repetitions. Source data are provided as a Source Data file.

Article Snippet: To measure Gα i -induced decreases in cAMP, we utilized a cAMP-dependent firefly luciferase suitable for Gα i signaling (GloSensor-22F; Promega; E2301) .

Techniques: Transfection, Staining, Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Control, Inhibition, Competitive Binding Assay