hek293 cells stably expressing the glosensor luciferase enzyme  (Promega)

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

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

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