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hek293 cells stably expressing the glosensor luciferase enzyme  (Promega)

 
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    Promega hek293 cells stably expressing the glosensor luciferase enzyme
    ( A ) <t>HEK293</t> cells stably expressing the GloSensor reporter monitoring the cAMP level were pretreated with either 25 μM Cmpd-6 or the control vehicle (DMSO) and stimulated with a serial dilution of either isoproterenol (ISO) or albuterol (Alb). The level of cAMP production induced by the endogenously expressed β 2 AR was determined as described in Methods. ( B ) <t>HEK293T</t> cells expressing all components for the Tango assays monitoring β-arrestin recruitment to the stably expressed β 2 V 2 R as described in Methods were pretreated with Cmpd-6 or DMSO and stimulated with the agonists as described for ( A ). The extent of β-arrestin recruitment was determined as described in Methods. The values in ( A and B ) were expressed as percentage of the ISO-stimulated maximal response in the DMSO-treated condition. ( C and D ) Isolated membranes from Expi293F cells transiently expressing the human β 2 AR were incubated together with either Cmpd-6 at indicated concentrations or DMSO, a serial dilution of the indicated agonist competitor, Alb ( C ) and ISO ( D ), and 60 pM [ 125 I]-cyanopindolol ( 125 I-CYP). The reaction was terminated, and 125 I-CYP bound to the receptor was read as described in Methods. Values were normalized to the percentage of the maximal 125 I-CYP binding level obtained in each of the Cmpd-6- and DMSO-treated conditions, with data points representing mean ± SEM, obtained from 4 ( A and B ) or 5 ( C and D ) independent experiments performed in duplicate. The shift of curves was expressed as fold changes in either EC 50 and B max ( A and B ) or IC 50 (C, D) values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for these shifts in each of the directions were performed using paired 2-tailed Student’s t tests. P values shown were * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control DMSO-treated condition.
    Hek293 Cells Stably Expressing The Glosensor Luciferase Enzyme, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hek293 cells stably expressing the glosensor luciferase enzyme - by Bioz Stars, 2026-11
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    1) Product Images from "Allosteric modulator potentiates β 2 AR agonist–promoted bronchoprotection in asthma models"

    Article Title: Allosteric modulator potentiates β 2 AR agonist–promoted bronchoprotection in asthma models

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI167337

    ( A ) HEK293 cells stably expressing the GloSensor reporter monitoring the cAMP level were pretreated with either 25 μM Cmpd-6 or the control vehicle (DMSO) and stimulated with a serial dilution of either isoproterenol (ISO) or albuterol (Alb). The level of cAMP production induced by the endogenously expressed β 2 AR was determined as described in Methods. ( B ) HEK293T cells expressing all components for the Tango assays monitoring β-arrestin recruitment to the stably expressed β 2 V 2 R as described in Methods were pretreated with Cmpd-6 or DMSO and stimulated with the agonists as described for ( A ). The extent of β-arrestin recruitment was determined as described in Methods. The values in ( A and B ) were expressed as percentage of the ISO-stimulated maximal response in the DMSO-treated condition. ( C and D ) Isolated membranes from Expi293F cells transiently expressing the human β 2 AR were incubated together with either Cmpd-6 at indicated concentrations or DMSO, a serial dilution of the indicated agonist competitor, Alb ( C ) and ISO ( D ), and 60 pM [ 125 I]-cyanopindolol ( 125 I-CYP). The reaction was terminated, and 125 I-CYP bound to the receptor was read as described in Methods. Values were normalized to the percentage of the maximal 125 I-CYP binding level obtained in each of the Cmpd-6- and DMSO-treated conditions, with data points representing mean ± SEM, obtained from 4 ( A and B ) or 5 ( C and D ) independent experiments performed in duplicate. The shift of curves was expressed as fold changes in either EC 50 and B max ( A and B ) or IC 50 (C, D) values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for these shifts in each of the directions were performed using paired 2-tailed Student’s t tests. P values shown were * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control DMSO-treated condition.
    Figure Legend Snippet: ( A ) HEK293 cells stably expressing the GloSensor reporter monitoring the cAMP level were pretreated with either 25 μM Cmpd-6 or the control vehicle (DMSO) and stimulated with a serial dilution of either isoproterenol (ISO) or albuterol (Alb). The level of cAMP production induced by the endogenously expressed β 2 AR was determined as described in Methods. ( B ) HEK293T cells expressing all components for the Tango assays monitoring β-arrestin recruitment to the stably expressed β 2 V 2 R as described in Methods were pretreated with Cmpd-6 or DMSO and stimulated with the agonists as described for ( A ). The extent of β-arrestin recruitment was determined as described in Methods. The values in ( A and B ) were expressed as percentage of the ISO-stimulated maximal response in the DMSO-treated condition. ( C and D ) Isolated membranes from Expi293F cells transiently expressing the human β 2 AR were incubated together with either Cmpd-6 at indicated concentrations or DMSO, a serial dilution of the indicated agonist competitor, Alb ( C ) and ISO ( D ), and 60 pM [ 125 I]-cyanopindolol ( 125 I-CYP). The reaction was terminated, and 125 I-CYP bound to the receptor was read as described in Methods. Values were normalized to the percentage of the maximal 125 I-CYP binding level obtained in each of the Cmpd-6- and DMSO-treated conditions, with data points representing mean ± SEM, obtained from 4 ( A and B ) or 5 ( C and D ) independent experiments performed in duplicate. The shift of curves was expressed as fold changes in either EC 50 and B max ( A and B ) or IC 50 (C, D) values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for these shifts in each of the directions were performed using paired 2-tailed Student’s t tests. P values shown were * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control DMSO-treated condition.

    Techniques Used: Stable Transfection, Expressing, Control, Serial Dilution, Isolation, Incubation, Binding Assay

    ( A ) The sequence alignment of amino acids composing the Cmpd-6 binding site between the human and murine β 2 ARs. Shaded (yellow) amino acids represent the most essential ones, phenylalanine-133 (F-133), substituted to valine (V) in the murine receptor, and lysine-141 (K-141), for Cmpd-6 binding to the β 2 AR. ( B ) The Cmpd-6 binding site in the human (left) and modeled murine (right) β 2 ARs shows the topographical molecular surface of F-133 (purple) and V-133 (cyan) on the transmembrane-3 (TM-3). Illustrations were created with the previously reported structure PDB-6N48 using the PyMOL program. ( C ) Radioligand competition binding was performed with isolated membranes from 293ExpiF cells transiently expressing either the WT or V133F mutant murine β 2 AR as described in and D. Curve fits were plotted with data sets obtained from 4 independent experiments done in duplicate. The shift of curves was expressed as fold changes in IC 50 values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for the shift in each of the WT and mutant receptor were performed using paired 2-tailed Student’s t tests. *** P adj < 0.001 compared with the DMSO-treated condition. ( D ) HEK293 cells stably expressing the GloSensor reporter were transiently transfected with one of the human WT, murine WT, and murine V133F mutant β 2 AR. After incubation with Cmpd-6 at 5 μM or DMSO vehicle, the cAMP level was monitored as described in Methods. Values were expressed as percentage of the isoproterenol-stimulated (ISO-stimulated) maximal response obtained as a control for comparable receptor expression in each transfection condition and represent mean ± SEM obtained from 4 independent experiments performed in duplicate. Statistical analyses were performed using 1-way ANOVA, repeated (related) measures with Tukey’s posthoc test. Adjusted ** P adj < 0.01.
    Figure Legend Snippet: ( A ) The sequence alignment of amino acids composing the Cmpd-6 binding site between the human and murine β 2 ARs. Shaded (yellow) amino acids represent the most essential ones, phenylalanine-133 (F-133), substituted to valine (V) in the murine receptor, and lysine-141 (K-141), for Cmpd-6 binding to the β 2 AR. ( B ) The Cmpd-6 binding site in the human (left) and modeled murine (right) β 2 ARs shows the topographical molecular surface of F-133 (purple) and V-133 (cyan) on the transmembrane-3 (TM-3). Illustrations were created with the previously reported structure PDB-6N48 using the PyMOL program. ( C ) Radioligand competition binding was performed with isolated membranes from 293ExpiF cells transiently expressing either the WT or V133F mutant murine β 2 AR as described in and D. Curve fits were plotted with data sets obtained from 4 independent experiments done in duplicate. The shift of curves was expressed as fold changes in IC 50 values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for the shift in each of the WT and mutant receptor were performed using paired 2-tailed Student’s t tests. *** P adj < 0.001 compared with the DMSO-treated condition. ( D ) HEK293 cells stably expressing the GloSensor reporter were transiently transfected with one of the human WT, murine WT, and murine V133F mutant β 2 AR. After incubation with Cmpd-6 at 5 μM or DMSO vehicle, the cAMP level was monitored as described in Methods. Values were expressed as percentage of the isoproterenol-stimulated (ISO-stimulated) maximal response obtained as a control for comparable receptor expression in each transfection condition and represent mean ± SEM obtained from 4 independent experiments performed in duplicate. Statistical analyses were performed using 1-way ANOVA, repeated (related) measures with Tukey’s posthoc test. Adjusted ** P adj < 0.01.

    Techniques Used: Sequencing, Binding Assay, Isolation, Expressing, Mutagenesis, Stable Transfection, Transfection, Incubation, Control

    ( A ) The sequence alignment of amino acids composing the Cmpd-6 binding site between the human and guinea pig β 2 ARs. Shaded (yellow) amino acids represent the most essential ones, phenylalanine-133 (F-133) and lysine-141 (K-141), for Cmpd-6 binding to the β 2 AR. ( B ) Radioligand competition binding was performed with isolated membranes from 293ExpiF cells transiently expressing the guinea pig β 2 AR as described in and D. Curve fits were plotted by a 1-site competition binding-log IC 50 curve fit (GraphPad Prism) with data sets obtained from 5 independent experiments done in duplicate. The shift of curves was expressed as fold changes in IC 50 values and statistically analyzed using paired 2-tailed Student’s t test between the highest concentration of Cmpd-6– and DMSO-treated conditions. ** P < 0.01. ( C ) Cmpd-6 was incubated for 20 minutes with HEK293 cells expressing the GloSensor reporter stably and the guinea pig β 2 AR transiently. The extent of cAMP generation was determined and normalized as described for D. The value represents mean ± SEM obtained from 4 independent experiments performed in duplicate. The lines indicate the level of Cmpd-6–induced cAMP production driven by overexpression of the human (black) and the murine (red) β 2 AR shown in D. ( D – F ) Radioligand competition binding was performed as essentially described above for ( B ) with multiple β 2 agonists as competitors: fenoterol (FEN; D ), albuterol (Alb; E ), and salmeterol (Salm; F ). Curve fits, the expression of the curve shift, and statistical analyses were also generated as described for ( B ) with data sets obtained from 4 independent experiments performed in duplicate. *** P < 0.001.
    Figure Legend Snippet: ( A ) The sequence alignment of amino acids composing the Cmpd-6 binding site between the human and guinea pig β 2 ARs. Shaded (yellow) amino acids represent the most essential ones, phenylalanine-133 (F-133) and lysine-141 (K-141), for Cmpd-6 binding to the β 2 AR. ( B ) Radioligand competition binding was performed with isolated membranes from 293ExpiF cells transiently expressing the guinea pig β 2 AR as described in and D. Curve fits were plotted by a 1-site competition binding-log IC 50 curve fit (GraphPad Prism) with data sets obtained from 5 independent experiments done in duplicate. The shift of curves was expressed as fold changes in IC 50 values and statistically analyzed using paired 2-tailed Student’s t test between the highest concentration of Cmpd-6– and DMSO-treated conditions. ** P < 0.01. ( C ) Cmpd-6 was incubated for 20 minutes with HEK293 cells expressing the GloSensor reporter stably and the guinea pig β 2 AR transiently. The extent of cAMP generation was determined and normalized as described for D. The value represents mean ± SEM obtained from 4 independent experiments performed in duplicate. The lines indicate the level of Cmpd-6–induced cAMP production driven by overexpression of the human (black) and the murine (red) β 2 AR shown in D. ( D – F ) Radioligand competition binding was performed as essentially described above for ( B ) with multiple β 2 agonists as competitors: fenoterol (FEN; D ), albuterol (Alb; E ), and salmeterol (Salm; F ). Curve fits, the expression of the curve shift, and statistical analyses were also generated as described for ( B ) with data sets obtained from 4 independent experiments performed in duplicate. *** P < 0.001.

    Techniques Used: Sequencing, Binding Assay, Isolation, Expressing, Concentration Assay, Incubation, Stable Transfection, Over Expression, Generated

    Related Articles

    Stable Transfection:

    Article Title: Unique Positive Cooperativity Between the β -Arrestin–Biased β -Blocker Carvedilol and a Small Molecule Positive Allosteric Modulator of the β 2-Adrenergic Receptor
    Article Snippet: Specific 35 S-GTP γ S binding for respective conditions was normalized to no ligand stimulation, expressed as fold over basal, and plotted as bar graphs in GraphPad Prism. .. cAMP production was monitored at endogenous β 2AR in HEK293 cells stably expressing the plasmid for GloSensor luciferase enzyme (Promega), a chemiluminescence-based cAMP biosensor. .. Cells were plated in 96-well, white clear-bottom plates at a density of ∼80,000 cells per well, and on the following day, chemiluminescence signals generated by the GloSensor luciferase were read using a CLARIOstar microplate reader (BMG Labtech, Cary, NC) as previously described ( ).

    Article Title: Allosteric modulator potentiates β2AR agonist–promoted bronchoprotection in asthma models
    Article Snippet: .. HEK293 cells stably expressing the GloSensor luciferase enzyme (Promega), in the absence or presence of transient transfection with each of the constructs for expressing the β2AR from different species, were plated at a density of approximately 80,000 cells on each well of a 96-well, white clear-bottom plate. .. At 20–24 hours after plating the cells, the GloSensor reagent (Promega) was prepared and added onto the cells in each well according to the manufacturer’s instructions.

    Expressing:

    Article Title: Unique Positive Cooperativity Between the β -Arrestin–Biased β -Blocker Carvedilol and a Small Molecule Positive Allosteric Modulator of the β 2-Adrenergic Receptor
    Article Snippet: Specific 35 S-GTP γ S binding for respective conditions was normalized to no ligand stimulation, expressed as fold over basal, and plotted as bar graphs in GraphPad Prism. .. cAMP production was monitored at endogenous β 2AR in HEK293 cells stably expressing the plasmid for GloSensor luciferase enzyme (Promega), a chemiluminescence-based cAMP biosensor. .. Cells were plated in 96-well, white clear-bottom plates at a density of ∼80,000 cells per well, and on the following day, chemiluminescence signals generated by the GloSensor luciferase were read using a CLARIOstar microplate reader (BMG Labtech, Cary, NC) as previously described ( ).

    Article Title: Allosteric modulator potentiates β2AR agonist–promoted bronchoprotection in asthma models
    Article Snippet: .. HEK293 cells stably expressing the GloSensor luciferase enzyme (Promega), in the absence or presence of transient transfection with each of the constructs for expressing the β2AR from different species, were plated at a density of approximately 80,000 cells on each well of a 96-well, white clear-bottom plate. .. At 20–24 hours after plating the cells, the GloSensor reagent (Promega) was prepared and added onto the cells in each well according to the manufacturer’s instructions.

    Plasmid Preparation:

    Article Title: Unique Positive Cooperativity Between the β -Arrestin–Biased β -Blocker Carvedilol and a Small Molecule Positive Allosteric Modulator of the β 2-Adrenergic Receptor
    Article Snippet: Specific 35 S-GTP γ S binding for respective conditions was normalized to no ligand stimulation, expressed as fold over basal, and plotted as bar graphs in GraphPad Prism. .. cAMP production was monitored at endogenous β 2AR in HEK293 cells stably expressing the plasmid for GloSensor luciferase enzyme (Promega), a chemiluminescence-based cAMP biosensor. .. Cells were plated in 96-well, white clear-bottom plates at a density of ∼80,000 cells per well, and on the following day, chemiluminescence signals generated by the GloSensor luciferase were read using a CLARIOstar microplate reader (BMG Labtech, Cary, NC) as previously described ( ).

    Luciferase:

    Article Title: Unique Positive Cooperativity Between the β -Arrestin–Biased β -Blocker Carvedilol and a Small Molecule Positive Allosteric Modulator of the β 2-Adrenergic Receptor
    Article Snippet: Specific 35 S-GTP γ S binding for respective conditions was normalized to no ligand stimulation, expressed as fold over basal, and plotted as bar graphs in GraphPad Prism. .. cAMP production was monitored at endogenous β 2AR in HEK293 cells stably expressing the plasmid for GloSensor luciferase enzyme (Promega), a chemiluminescence-based cAMP biosensor. .. Cells were plated in 96-well, white clear-bottom plates at a density of ∼80,000 cells per well, and on the following day, chemiluminescence signals generated by the GloSensor luciferase were read using a CLARIOstar microplate reader (BMG Labtech, Cary, NC) as previously described ( ).

    Article Title: Allosteric modulator potentiates β2AR agonist–promoted bronchoprotection in asthma models
    Article Snippet: .. HEK293 cells stably expressing the GloSensor luciferase enzyme (Promega), in the absence or presence of transient transfection with each of the constructs for expressing the β2AR from different species, were plated at a density of approximately 80,000 cells on each well of a 96-well, white clear-bottom plate. .. At 20–24 hours after plating the cells, the GloSensor reagent (Promega) was prepared and added onto the cells in each well according to the manufacturer’s instructions.

    Transfection:

    Article Title: Allosteric modulator potentiates β2AR agonist–promoted bronchoprotection in asthma models
    Article Snippet: .. HEK293 cells stably expressing the GloSensor luciferase enzyme (Promega), in the absence or presence of transient transfection with each of the constructs for expressing the β2AR from different species, were plated at a density of approximately 80,000 cells on each well of a 96-well, white clear-bottom plate. .. At 20–24 hours after plating the cells, the GloSensor reagent (Promega) was prepared and added onto the cells in each well according to the manufacturer’s instructions.

    Construct:

    Article Title: Allosteric modulator potentiates β2AR agonist–promoted bronchoprotection in asthma models
    Article Snippet: .. HEK293 cells stably expressing the GloSensor luciferase enzyme (Promega), in the absence or presence of transient transfection with each of the constructs for expressing the β2AR from different species, were plated at a density of approximately 80,000 cells on each well of a 96-well, white clear-bottom plate. .. At 20–24 hours after plating the cells, the GloSensor reagent (Promega) was prepared and added onto the cells in each well according to the manufacturer’s instructions.



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    hek293 cells stably expressing the glosensor luciferase enzyme  (Promega)
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    ( A ) <t>HEK293</t> cells stably expressing the GloSensor reporter monitoring the cAMP level were pretreated with either 25 μM Cmpd-6 or the control vehicle (DMSO) and stimulated with a serial dilution of either isoproterenol (ISO) or albuterol (Alb). The level of cAMP production induced by the endogenously expressed β 2 AR was determined as described in Methods. ( B ) <t>HEK293T</t> cells expressing all components for the Tango assays monitoring β-arrestin recruitment to the stably expressed β 2 V 2 R as described in Methods were pretreated with Cmpd-6 or DMSO and stimulated with the agonists as described for ( A ). The extent of β-arrestin recruitment was determined as described in Methods. The values in ( A and B ) were expressed as percentage of the ISO-stimulated maximal response in the DMSO-treated condition. ( C and D ) Isolated membranes from Expi293F cells transiently expressing the human β 2 AR were incubated together with either Cmpd-6 at indicated concentrations or DMSO, a serial dilution of the indicated agonist competitor, Alb ( C ) and ISO ( D ), and 60 pM [ 125 I]-cyanopindolol ( 125 I-CYP). The reaction was terminated, and 125 I-CYP bound to the receptor was read as described in Methods. Values were normalized to the percentage of the maximal 125 I-CYP binding level obtained in each of the Cmpd-6- and DMSO-treated conditions, with data points representing mean ± SEM, obtained from 4 ( A and B ) or 5 ( C and D ) independent experiments performed in duplicate. The shift of curves was expressed as fold changes in either EC 50 and B max ( A and B ) or IC 50 (C, D) values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for these shifts in each of the directions were performed using paired 2-tailed Student’s t tests. P values shown were * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control DMSO-treated condition.
    Hek293 Cells Stably Expressing The Glosensor Luciferase Enzyme, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    glosensor luciferase enzyme  (Promega)
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    Promega glosensor luciferase enzyme
    ( A ) <t>HEK293</t> cells stably expressing the GloSensor reporter monitoring the cAMP level were pretreated with either 25 μM Cmpd-6 or the control vehicle (DMSO) and stimulated with a serial dilution of either isoproterenol (ISO) or albuterol (Alb). The level of cAMP production induced by the endogenously expressed β 2 AR was determined as described in Methods. ( B ) <t>HEK293T</t> cells expressing all components for the Tango assays monitoring β-arrestin recruitment to the stably expressed β 2 V 2 R as described in Methods were pretreated with Cmpd-6 or DMSO and stimulated with the agonists as described for ( A ). The extent of β-arrestin recruitment was determined as described in Methods. The values in ( A and B ) were expressed as percentage of the ISO-stimulated maximal response in the DMSO-treated condition. ( C and D ) Isolated membranes from Expi293F cells transiently expressing the human β 2 AR were incubated together with either Cmpd-6 at indicated concentrations or DMSO, a serial dilution of the indicated agonist competitor, Alb ( C ) and ISO ( D ), and 60 pM [ 125 I]-cyanopindolol ( 125 I-CYP). The reaction was terminated, and 125 I-CYP bound to the receptor was read as described in Methods. Values were normalized to the percentage of the maximal 125 I-CYP binding level obtained in each of the Cmpd-6- and DMSO-treated conditions, with data points representing mean ± SEM, obtained from 4 ( A and B ) or 5 ( C and D ) independent experiments performed in duplicate. The shift of curves was expressed as fold changes in either EC 50 and B max ( A and B ) or IC 50 (C, D) values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for these shifts in each of the directions were performed using paired 2-tailed Student’s t tests. P values shown were * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control DMSO-treated condition.
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    ( A ) HEK293 cells stably expressing the GloSensor reporter monitoring the cAMP level were pretreated with either 25 μM Cmpd-6 or the control vehicle (DMSO) and stimulated with a serial dilution of either isoproterenol (ISO) or albuterol (Alb). The level of cAMP production induced by the endogenously expressed β 2 AR was determined as described in Methods. ( B ) HEK293T cells expressing all components for the Tango assays monitoring β-arrestin recruitment to the stably expressed β 2 V 2 R as described in Methods were pretreated with Cmpd-6 or DMSO and stimulated with the agonists as described for ( A ). The extent of β-arrestin recruitment was determined as described in Methods. The values in ( A and B ) were expressed as percentage of the ISO-stimulated maximal response in the DMSO-treated condition. ( C and D ) Isolated membranes from Expi293F cells transiently expressing the human β 2 AR were incubated together with either Cmpd-6 at indicated concentrations or DMSO, a serial dilution of the indicated agonist competitor, Alb ( C ) and ISO ( D ), and 60 pM [ 125 I]-cyanopindolol ( 125 I-CYP). The reaction was terminated, and 125 I-CYP bound to the receptor was read as described in Methods. Values were normalized to the percentage of the maximal 125 I-CYP binding level obtained in each of the Cmpd-6- and DMSO-treated conditions, with data points representing mean ± SEM, obtained from 4 ( A and B ) or 5 ( C and D ) independent experiments performed in duplicate. The shift of curves was expressed as fold changes in either EC 50 and B max ( A and B ) or IC 50 (C, D) values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for these shifts in each of the directions were performed using paired 2-tailed Student’s t tests. P values shown were * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control DMSO-treated condition.

    Journal: The Journal of Clinical Investigation

    Article Title: Allosteric modulator potentiates β 2 AR agonist–promoted bronchoprotection in asthma models

    doi: 10.1172/JCI167337

    Figure Lengend Snippet: ( A ) HEK293 cells stably expressing the GloSensor reporter monitoring the cAMP level were pretreated with either 25 μM Cmpd-6 or the control vehicle (DMSO) and stimulated with a serial dilution of either isoproterenol (ISO) or albuterol (Alb). The level of cAMP production induced by the endogenously expressed β 2 AR was determined as described in Methods. ( B ) HEK293T cells expressing all components for the Tango assays monitoring β-arrestin recruitment to the stably expressed β 2 V 2 R as described in Methods were pretreated with Cmpd-6 or DMSO and stimulated with the agonists as described for ( A ). The extent of β-arrestin recruitment was determined as described in Methods. The values in ( A and B ) were expressed as percentage of the ISO-stimulated maximal response in the DMSO-treated condition. ( C and D ) Isolated membranes from Expi293F cells transiently expressing the human β 2 AR were incubated together with either Cmpd-6 at indicated concentrations or DMSO, a serial dilution of the indicated agonist competitor, Alb ( C ) and ISO ( D ), and 60 pM [ 125 I]-cyanopindolol ( 125 I-CYP). The reaction was terminated, and 125 I-CYP bound to the receptor was read as described in Methods. Values were normalized to the percentage of the maximal 125 I-CYP binding level obtained in each of the Cmpd-6- and DMSO-treated conditions, with data points representing mean ± SEM, obtained from 4 ( A and B ) or 5 ( C and D ) independent experiments performed in duplicate. The shift of curves was expressed as fold changes in either EC 50 and B max ( A and B ) or IC 50 (C, D) values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for these shifts in each of the directions were performed using paired 2-tailed Student’s t tests. P values shown were * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control DMSO-treated condition.

    Article Snippet: HEK293 cells stably expressing the GloSensor luciferase enzyme (Promega), in the absence or presence of transient transfection with each of the constructs for expressing the β 2 AR from different species, were plated at a density of approximately 80,000 cells on each well of a 96-well, white clear-bottom plate.

    Techniques: Stable Transfection, Expressing, Control, Serial Dilution, Isolation, Incubation, Binding Assay

    ( A ) The sequence alignment of amino acids composing the Cmpd-6 binding site between the human and murine β 2 ARs. Shaded (yellow) amino acids represent the most essential ones, phenylalanine-133 (F-133), substituted to valine (V) in the murine receptor, and lysine-141 (K-141), for Cmpd-6 binding to the β 2 AR. ( B ) The Cmpd-6 binding site in the human (left) and modeled murine (right) β 2 ARs shows the topographical molecular surface of F-133 (purple) and V-133 (cyan) on the transmembrane-3 (TM-3). Illustrations were created with the previously reported structure PDB-6N48 using the PyMOL program. ( C ) Radioligand competition binding was performed with isolated membranes from 293ExpiF cells transiently expressing either the WT or V133F mutant murine β 2 AR as described in and D. Curve fits were plotted with data sets obtained from 4 independent experiments done in duplicate. The shift of curves was expressed as fold changes in IC 50 values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for the shift in each of the WT and mutant receptor were performed using paired 2-tailed Student’s t tests. *** P adj < 0.001 compared with the DMSO-treated condition. ( D ) HEK293 cells stably expressing the GloSensor reporter were transiently transfected with one of the human WT, murine WT, and murine V133F mutant β 2 AR. After incubation with Cmpd-6 at 5 μM or DMSO vehicle, the cAMP level was monitored as described in Methods. Values were expressed as percentage of the isoproterenol-stimulated (ISO-stimulated) maximal response obtained as a control for comparable receptor expression in each transfection condition and represent mean ± SEM obtained from 4 independent experiments performed in duplicate. Statistical analyses were performed using 1-way ANOVA, repeated (related) measures with Tukey’s posthoc test. Adjusted ** P adj < 0.01.

    Journal: The Journal of Clinical Investigation

    Article Title: Allosteric modulator potentiates β 2 AR agonist–promoted bronchoprotection in asthma models

    doi: 10.1172/JCI167337

    Figure Lengend Snippet: ( A ) The sequence alignment of amino acids composing the Cmpd-6 binding site between the human and murine β 2 ARs. Shaded (yellow) amino acids represent the most essential ones, phenylalanine-133 (F-133), substituted to valine (V) in the murine receptor, and lysine-141 (K-141), for Cmpd-6 binding to the β 2 AR. ( B ) The Cmpd-6 binding site in the human (left) and modeled murine (right) β 2 ARs shows the topographical molecular surface of F-133 (purple) and V-133 (cyan) on the transmembrane-3 (TM-3). Illustrations were created with the previously reported structure PDB-6N48 using the PyMOL program. ( C ) Radioligand competition binding was performed with isolated membranes from 293ExpiF cells transiently expressing either the WT or V133F mutant murine β 2 AR as described in and D. Curve fits were plotted with data sets obtained from 4 independent experiments done in duplicate. The shift of curves was expressed as fold changes in IC 50 values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for the shift in each of the WT and mutant receptor were performed using paired 2-tailed Student’s t tests. *** P adj < 0.001 compared with the DMSO-treated condition. ( D ) HEK293 cells stably expressing the GloSensor reporter were transiently transfected with one of the human WT, murine WT, and murine V133F mutant β 2 AR. After incubation with Cmpd-6 at 5 μM or DMSO vehicle, the cAMP level was monitored as described in Methods. Values were expressed as percentage of the isoproterenol-stimulated (ISO-stimulated) maximal response obtained as a control for comparable receptor expression in each transfection condition and represent mean ± SEM obtained from 4 independent experiments performed in duplicate. Statistical analyses were performed using 1-way ANOVA, repeated (related) measures with Tukey’s posthoc test. Adjusted ** P adj < 0.01.

    Article Snippet: HEK293 cells stably expressing the GloSensor luciferase enzyme (Promega), in the absence or presence of transient transfection with each of the constructs for expressing the β 2 AR from different species, were plated at a density of approximately 80,000 cells on each well of a 96-well, white clear-bottom plate.

    Techniques: Sequencing, Binding Assay, Isolation, Expressing, Mutagenesis, Stable Transfection, Transfection, Incubation, Control

    ( A ) The sequence alignment of amino acids composing the Cmpd-6 binding site between the human and guinea pig β 2 ARs. Shaded (yellow) amino acids represent the most essential ones, phenylalanine-133 (F-133) and lysine-141 (K-141), for Cmpd-6 binding to the β 2 AR. ( B ) Radioligand competition binding was performed with isolated membranes from 293ExpiF cells transiently expressing the guinea pig β 2 AR as described in and D. Curve fits were plotted by a 1-site competition binding-log IC 50 curve fit (GraphPad Prism) with data sets obtained from 5 independent experiments done in duplicate. The shift of curves was expressed as fold changes in IC 50 values and statistically analyzed using paired 2-tailed Student’s t test between the highest concentration of Cmpd-6– and DMSO-treated conditions. ** P < 0.01. ( C ) Cmpd-6 was incubated for 20 minutes with HEK293 cells expressing the GloSensor reporter stably and the guinea pig β 2 AR transiently. The extent of cAMP generation was determined and normalized as described for D. The value represents mean ± SEM obtained from 4 independent experiments performed in duplicate. The lines indicate the level of Cmpd-6–induced cAMP production driven by overexpression of the human (black) and the murine (red) β 2 AR shown in D. ( D – F ) Radioligand competition binding was performed as essentially described above for ( B ) with multiple β 2 agonists as competitors: fenoterol (FEN; D ), albuterol (Alb; E ), and salmeterol (Salm; F ). Curve fits, the expression of the curve shift, and statistical analyses were also generated as described for ( B ) with data sets obtained from 4 independent experiments performed in duplicate. *** P < 0.001.

    Journal: The Journal of Clinical Investigation

    Article Title: Allosteric modulator potentiates β 2 AR agonist–promoted bronchoprotection in asthma models

    doi: 10.1172/JCI167337

    Figure Lengend Snippet: ( A ) The sequence alignment of amino acids composing the Cmpd-6 binding site between the human and guinea pig β 2 ARs. Shaded (yellow) amino acids represent the most essential ones, phenylalanine-133 (F-133) and lysine-141 (K-141), for Cmpd-6 binding to the β 2 AR. ( B ) Radioligand competition binding was performed with isolated membranes from 293ExpiF cells transiently expressing the guinea pig β 2 AR as described in and D. Curve fits were plotted by a 1-site competition binding-log IC 50 curve fit (GraphPad Prism) with data sets obtained from 5 independent experiments done in duplicate. The shift of curves was expressed as fold changes in IC 50 values and statistically analyzed using paired 2-tailed Student’s t test between the highest concentration of Cmpd-6– and DMSO-treated conditions. ** P < 0.01. ( C ) Cmpd-6 was incubated for 20 minutes with HEK293 cells expressing the GloSensor reporter stably and the guinea pig β 2 AR transiently. The extent of cAMP generation was determined and normalized as described for D. The value represents mean ± SEM obtained from 4 independent experiments performed in duplicate. The lines indicate the level of Cmpd-6–induced cAMP production driven by overexpression of the human (black) and the murine (red) β 2 AR shown in D. ( D – F ) Radioligand competition binding was performed as essentially described above for ( B ) with multiple β 2 agonists as competitors: fenoterol (FEN; D ), albuterol (Alb; E ), and salmeterol (Salm; F ). Curve fits, the expression of the curve shift, and statistical analyses were also generated as described for ( B ) with data sets obtained from 4 independent experiments performed in duplicate. *** P < 0.001.

    Article Snippet: HEK293 cells stably expressing the GloSensor luciferase enzyme (Promega), in the absence or presence of transient transfection with each of the constructs for expressing the β 2 AR from different species, were plated at a density of approximately 80,000 cells on each well of a 96-well, white clear-bottom plate.

    Techniques: Sequencing, Binding Assay, Isolation, Expressing, Concentration Assay, Incubation, Stable Transfection, Over Expression, Generated