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isoproterenol  (Tocris)


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    Structured Review

    Tocris isoproterenol
    FIG. 2. Co-internalization of 1AR with 2AAR. HA-2AAR and FLAG-1AR were expressed either separately (solid bars) or together (striped bars) in HEK-293 cells. The internalization of 2AAR (A) and 1AR (B) was examined using a luminometer-based assay following 10-min stimulations with the -adrenergic agonist <t>isoproterenol</t> (Iso; 10 m), the 2-adrenergic agonist UK 14,304 (UK; 10 M), or a combina- tion of the two agonists together. As shown in A, 2AAR exhibited 15% internalization in response to UK stimulation but no significant inter- nalization in response to isoproterenol under any condition. Conversely, as shown in B, 1AR exhibited 25–30% internalization in response to isoproterenol but also exhibited 15% internalization in response to stimulation with UK. This effect was only observed, however, when 2AAR was coexpressed (** indicates significantly different from 1AR alone, p 0.01). These data suggest that 1AR can co-internalize with agonist-activated 2AAR. The bars and error bars represent the means S.E. for 4–5 independent experiments for each condition, with each experiment being performed in triplicate.
    Isoproterenol, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Heterodimerization of α2A- and β1-Adrenergic Receptors"

    Article Title: Heterodimerization of α2A- and β1-Adrenergic Receptors

    Journal: Journal of Biological Chemistry

    doi: 10.1074/jbc.m207968200

    FIG. 2. Co-internalization of 1AR with 2AAR. HA-2AAR and FLAG-1AR were expressed either separately (solid bars) or together (striped bars) in HEK-293 cells. The internalization of 2AAR (A) and 1AR (B) was examined using a luminometer-based assay following 10-min stimulations with the -adrenergic agonist isoproterenol (Iso; 10 m), the 2-adrenergic agonist UK 14,304 (UK; 10 M), or a combina- tion of the two agonists together. As shown in A, 2AAR exhibited 15% internalization in response to UK stimulation but no significant inter- nalization in response to isoproterenol under any condition. Conversely, as shown in B, 1AR exhibited 25–30% internalization in response to isoproterenol but also exhibited 15% internalization in response to stimulation with UK. This effect was only observed, however, when 2AAR was coexpressed (** indicates significantly different from 1AR alone, p 0.01). These data suggest that 1AR can co-internalize with agonist-activated 2AAR. The bars and error bars represent the means S.E. for 4–5 independent experiments for each condition, with each experiment being performed in triplicate.
    Figure Legend Snippet: FIG. 2. Co-internalization of 1AR with 2AAR. HA-2AAR and FLAG-1AR were expressed either separately (solid bars) or together (striped bars) in HEK-293 cells. The internalization of 2AAR (A) and 1AR (B) was examined using a luminometer-based assay following 10-min stimulations with the -adrenergic agonist isoproterenol (Iso; 10 m), the 2-adrenergic agonist UK 14,304 (UK; 10 M), or a combina- tion of the two agonists together. As shown in A, 2AAR exhibited 15% internalization in response to UK stimulation but no significant inter- nalization in response to isoproterenol under any condition. Conversely, as shown in B, 1AR exhibited 25–30% internalization in response to isoproterenol but also exhibited 15% internalization in response to stimulation with UK. This effect was only observed, however, when 2AAR was coexpressed (** indicates significantly different from 1AR alone, p 0.01). These data suggest that 1AR can co-internalize with agonist-activated 2AAR. The bars and error bars represent the means S.E. for 4–5 independent experiments for each condition, with each experiment being performed in triplicate.

    Techniques Used:

    FIG. 3. Immunofluorescence confocal microscopy reveals ago- nist-promoted co-internalization of 2A- and 1-adrenergic re- ceptors. HA-2AAR (red) and FLAG-1AR (green) were co-transfected into HEK-293 cells and visualized using secondary antibodies coupled to rhodamine and FITC, respectively. In the absence of agonist stimu- lation, immunostaining for both receptors was found predominantly in the plasma membrane (A–C). Stimulation with isoproterenol (Iso) for 10 min induced significant mobilization of 1AR inside the cell (D) but had no significant effect on the subcellular distribution of 2AAR (E and F). Stimulation with UK 14,034 (UK), in contrast, resulted in significant internalization of both 2AAR (H) and 1AR (G) and marked co-local- ization of the two receptors in intracellular regions (I, with co-localiza- tion indicated in yellow). The specificity of staining was determined in control (Con) experiments using both untransfected and transfected cells incubated in the absence and presence of the relevant primary antibodies. These data are representative of 3–5 experiments for each condition.
    Figure Legend Snippet: FIG. 3. Immunofluorescence confocal microscopy reveals ago- nist-promoted co-internalization of 2A- and 1-adrenergic re- ceptors. HA-2AAR (red) and FLAG-1AR (green) were co-transfected into HEK-293 cells and visualized using secondary antibodies coupled to rhodamine and FITC, respectively. In the absence of agonist stimu- lation, immunostaining for both receptors was found predominantly in the plasma membrane (A–C). Stimulation with isoproterenol (Iso) for 10 min induced significant mobilization of 1AR inside the cell (D) but had no significant effect on the subcellular distribution of 2AAR (E and F). Stimulation with UK 14,034 (UK), in contrast, resulted in significant internalization of both 2AAR (H) and 1AR (G) and marked co-local- ization of the two receptors in intracellular regions (I, with co-localiza- tion indicated in yellow). The specificity of staining was determined in control (Con) experiments using both untransfected and transfected cells incubated in the absence and presence of the relevant primary antibodies. These data are representative of 3–5 experiments for each condition.

    Techniques Used: Immunofluorescence, Confocal Microscopy, Transfection, Immunostaining, Clinical Proteomics, Membrane, Staining, Control, Incubation

    FIG. 5. Coexpression of 1AR with 2AAR alters the potency of isoproterenol-induced stimulation of adenylyl cyclase. HEK-293 cells were transfected with either 1AR alone (filled squares, solid line) or 1AR/2AAR (open triangles, dotted line). Expression levels of the 1AR were identical for the two transfection conditions, as assessed by Western blot. The cells were stimulated with increasing concentrations of isoproterenol, and agonist-induced rises in cellular cyclic AMP were quantified. The maximal extent of cyclic AMP produced in the 1AR/2AAR cells was 106 8% of that produced in the cells trans- fected with only 1AR. The EC50 for isoproterenol stimulation of 1AR alone was 0.16 0.02 nM, as compared with 0.68 0.17 for 1AR/ 2AAR (significantly different from 1AR alone, p 0.01). The points and error bars represent the means and S.E. values for four independ- ent determinations.
    Figure Legend Snippet: FIG. 5. Coexpression of 1AR with 2AAR alters the potency of isoproterenol-induced stimulation of adenylyl cyclase. HEK-293 cells were transfected with either 1AR alone (filled squares, solid line) or 1AR/2AAR (open triangles, dotted line). Expression levels of the 1AR were identical for the two transfection conditions, as assessed by Western blot. The cells were stimulated with increasing concentrations of isoproterenol, and agonist-induced rises in cellular cyclic AMP were quantified. The maximal extent of cyclic AMP produced in the 1AR/2AAR cells was 106 8% of that produced in the cells trans- fected with only 1AR. The EC50 for isoproterenol stimulation of 1AR alone was 0.16 0.02 nM, as compared with 0.68 0.17 for 1AR/ 2AAR (significantly different from 1AR alone, p 0.01). The points and error bars represent the means and S.E. values for four independ- ent determinations.

    Techniques Used: Transfection, Expressing, Western Blot, Produced

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    Article Title: The mGlu 5 Receptor Protomer-Mediated Dopamine D 2 Receptor Trans-Inhibition Is Dependent on the Adenosine A 2A Receptor Protomer: Implications for Parkinson's Disease.
    Article Snippet: Also, isobutyl-1-methylxanthine (IBMX) and 4-(3-butoxy-4-methoxybenzyl) imidazolidone (Ro 20-1724) were purchased from Tocris Bioscience (Bristol, UK).

    Incubation:

    Article Title: Compounds and methods of treating retinal degeneration
    Article Snippet: .. After 24 h, cells were washed with Krebs Ringer bicarbonate buffer containing glucose (KRBG), and incubated with KRBG buffer containing 100 μM cAMP specific phosphodiesterase inhibitor, Ro 20-1724 (Tocris, UK) at RT for 10 min Under dim red light, cells then were treated with 25 μL of 6× its final concentration of forskolin (final 20 μM) followed by addition of 25 μL 6× final concentration of 9-cis-retinal, YC-001 or 9-cis-retinal and YC-001 together. ..

    Concentration Assay:

    Article Title: Compounds and methods of treating retinal degeneration
    Article Snippet: .. After 24 h, cells were washed with Krebs Ringer bicarbonate buffer containing glucose (KRBG), and incubated with KRBG buffer containing 100 μM cAMP specific phosphodiesterase inhibitor, Ro 20-1724 (Tocris, UK) at RT for 10 min Under dim red light, cells then were treated with 25 μL of 6× its final concentration of forskolin (final 20 μM) followed by addition of 25 μL 6× final concentration of 9-cis-retinal, YC-001 or 9-cis-retinal and YC-001 together. ..



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    PKA inhibition rescues the mI AHP deficits in pyramidal tract neurons from Itgb3 knockout mice. ( A ) Proteomic analysis indicates that the Tau phosphorylation site Ser202 responds differentially to PKA activation (Forskolin, 25 μM, combined with 50 µM of the phosphodiesterase inhibitor Ro 20-1724) and inhibition (cAMPS-Rp, 100 μM) in wild-type (WT) and Itgb3 knockout (KO) prefrontal cortex slices ( n = 3–5 per condition; r = Pearson correlation coefficient). ( B ) Western blots for pTau (Ser202/Thr205) and Tau from WT and Itgb3 KO cortical slices with or without treatment with the PKA inhibitor cAMPS-Rp (100 μM). ( C ) Quantification of experiments as in B , indicating increased basal phosphorylation levels of Tau at Ser202/Thr205 in prefrontal cortex slices from Itgb3 KO mice and opposite effects of cAMPS-Rp in the two genotypes ( n = 5 mice per genotype; * P = 0.03, ** P < 0.01, two-way ANOVA followed by Fisher’s least significant difference post-test). ( D ) Apamin-sensitive tail currents in pyramidal tract (PT) and intratelencephalic (IT) neurons from WT and Itgb3 KO mice recorded as in but with the addition of cAMPS-Rp in the intracellular solution (100 μM). Insets : Digital subtraction of the traces before and after application of apamin. Scale bars ( insets ): 50 pA and 100 ms. ( E ) Amplitude ( left ), peak time ( middle ) and weighted decay time constant ( right ) of the apamin-sensitive mI AHP for experiments as in D . In the presence of cAMPS-Rp, the mI AHP in KO PT neurons is as large as in WT PT neurons ( n = 6 neurons per group; *** P < 0.001, two-way ANOVA followed by Sidak’s post-test). In the box and whisker plots, the horizontal line indicates the median, the box represents the interquartile range and the whiskers extend from the 10th to 90th percentiles. See also and . mI AHP = medium afterhyperpolarization current.

    Journal: Brain

    Article Title: Dysregulated cortical excitability and tau phosphorylation in a β3 integrin mouse model of autism

    doi: 10.1093/brain/awaf089

    Figure Lengend Snippet: PKA inhibition rescues the mI AHP deficits in pyramidal tract neurons from Itgb3 knockout mice. ( A ) Proteomic analysis indicates that the Tau phosphorylation site Ser202 responds differentially to PKA activation (Forskolin, 25 μM, combined with 50 µM of the phosphodiesterase inhibitor Ro 20-1724) and inhibition (cAMPS-Rp, 100 μM) in wild-type (WT) and Itgb3 knockout (KO) prefrontal cortex slices ( n = 3–5 per condition; r = Pearson correlation coefficient). ( B ) Western blots for pTau (Ser202/Thr205) and Tau from WT and Itgb3 KO cortical slices with or without treatment with the PKA inhibitor cAMPS-Rp (100 μM). ( C ) Quantification of experiments as in B , indicating increased basal phosphorylation levels of Tau at Ser202/Thr205 in prefrontal cortex slices from Itgb3 KO mice and opposite effects of cAMPS-Rp in the two genotypes ( n = 5 mice per genotype; * P = 0.03, ** P < 0.01, two-way ANOVA followed by Fisher’s least significant difference post-test). ( D ) Apamin-sensitive tail currents in pyramidal tract (PT) and intratelencephalic (IT) neurons from WT and Itgb3 KO mice recorded as in but with the addition of cAMPS-Rp in the intracellular solution (100 μM). Insets : Digital subtraction of the traces before and after application of apamin. Scale bars ( insets ): 50 pA and 100 ms. ( E ) Amplitude ( left ), peak time ( middle ) and weighted decay time constant ( right ) of the apamin-sensitive mI AHP for experiments as in D . In the presence of cAMPS-Rp, the mI AHP in KO PT neurons is as large as in WT PT neurons ( n = 6 neurons per group; *** P < 0.001, two-way ANOVA followed by Sidak’s post-test). In the box and whisker plots, the horizontal line indicates the median, the box represents the interquartile range and the whiskers extend from the 10th to 90th percentiles. See also and . mI AHP = medium afterhyperpolarization current.

    Article Snippet: Brain slices were divided into three experimental groups: (i) Sham: slices kept at 30°C for 30 min; (ii) Forskolin: slices treated with the adenylyl cyclase activator Forskolin (25 μM; Cat. No. 1099, Tocris) and the phosphodiesterase inhibitor Ro 20-1724 (50 μM; Cat. No. 0415, Tocris) for 15 min at 30°C; and (iii) cAMPS-Rp: slices treated with the PKA inhibitor cAMPS-Rp (100 μM; Cat. No. A165, Sigma) for 30 min at 30°C.

    Techniques: Inhibition, Knock-Out, Phospho-proteomics, Activation Assay, Western Blot, Whisker Assay