brefeldin a  (Tocris)

 
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  • 98
    Name:
    Brefeldin A
    Description:
    Disrupts protein translocation to Golgi
    Catalog Number:
    1231
    Price:
    None
    Purity:
    ≥98% (HPLC)
    Category:
    Translocation Exocytosis Endocytosis Signal Transduction Cell Biology
    Formula:
    1,6,7,8,9,11aβ,12,13,14,14αa-Decahydro-1β,13α-dihydroxy-6β-methyl-4H-cyclopent(f)oxacyclotridecin-4-one
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    Structured Review

    Tocris brefeldin a
    Brefeldin A
    Disrupts protein translocation to Golgi
    https://www.bioz.com/result/brefeldin a/product/Tocris
    Average 98 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    brefeldin a - by Bioz Stars, 2020-11
    98/100 stars

    Images

    1) Product Images from "Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors"

    Article Title: Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29262-4

    CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).
    Figure Legend Snippet: CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).

    Techniques Used: Purification, Selection, Cell Culture, Staining, Flow Cytometry, Cytometry

    2) Product Images from "Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study"

    Article Title: Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13901

    Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P
    Figure Legend Snippet: Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P

    Techniques Used: Functional Assay, Inhibition

    Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).
    Figure Legend Snippet: Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).

    Techniques Used: Functional Assay, Blocking Assay

    3) Product Images from "Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed ?2-Adrenergic Receptors"

    Article Title: Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed ?2-Adrenergic Receptors

    Journal: Scientific Reports

    doi: 10.1038/srep03828

    The effect of Epac inhibition on the DMR of different ligands. (a) DMR heatmap of AR ligands in the subclone A and in the 20 μM brefeldin A (BFA)- and RNAi Epac1-treated cells. The real responses of all ligands in the untreated cells were used for visualizing their DMR characteristics, while the treatment-induced net changes were used for better visualization of the effect of Epac inhibition. Only ligands that gave rise to a DMR of > 40 pm or an Epac inhibition-induced net change of > 40 pm were included in this analysis. (b–i) The real-time DMR of different ligands in cells without (control) or with brefeldin A- or Epac1 RNAi pretreatment: (b) epinephrine; (c) UK14,304; (d) isoproterenol; (e) Isoetharine; (f) cimaterol; (g) clenbuterol; (h) salbutamol; (i) betaxolol. All ligands were profiled at 10 μM. Data represents mean ± s.d. (n = 4).
    Figure Legend Snippet: The effect of Epac inhibition on the DMR of different ligands. (a) DMR heatmap of AR ligands in the subclone A and in the 20 μM brefeldin A (BFA)- and RNAi Epac1-treated cells. The real responses of all ligands in the untreated cells were used for visualizing their DMR characteristics, while the treatment-induced net changes were used for better visualization of the effect of Epac inhibition. Only ligands that gave rise to a DMR of > 40 pm or an Epac inhibition-induced net change of > 40 pm were included in this analysis. (b–i) The real-time DMR of different ligands in cells without (control) or with brefeldin A- or Epac1 RNAi pretreatment: (b) epinephrine; (c) UK14,304; (d) isoproterenol; (e) Isoetharine; (f) cimaterol; (g) clenbuterol; (h) salbutamol; (i) betaxolol. All ligands were profiled at 10 μM. Data represents mean ± s.d. (n = 4).

    Techniques Used: Inhibition

    4) Product Images from "Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study"

    Article Title: Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13901

    Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P
    Figure Legend Snippet: Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P

    Techniques Used: Functional Assay, Inhibition

    Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).
    Figure Legend Snippet: Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).

    Techniques Used: Functional Assay, Blocking Assay

    5) Product Images from "Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study"

    Article Title: Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13901

    Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P
    Figure Legend Snippet: Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P

    Techniques Used: Functional Assay, Inhibition

    Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).
    Figure Legend Snippet: Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).

    Techniques Used: Functional Assay, Blocking Assay

    6) Product Images from "Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study"

    Article Title: Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13901

    Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P
    Figure Legend Snippet: Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P

    Techniques Used: Functional Assay, Inhibition

    Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).
    Figure Legend Snippet: Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).

    Techniques Used: Functional Assay, Blocking Assay

    7) Product Images from "Concerted actions of NHERF2 and WNK4 in regulating TRPV5"

    Article Title: Concerted actions of NHERF2 and WNK4 in regulating TRPV5

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2010.12.095

    NHERF2 did not alter the forward trafficking of TRPV5 but rendered TRPV5 more stable at the plasma membrane in the presence or absence of WNK4. A . NHERF2 didn’t improve the lack of robust response of N -glycosylation defective N358Q mutant of TRPV5 to the regulation by WNK4 (n = 14). B . Assessment of WNK4 and NHERF2 on TRPV5 forward trafficking. Oocytes injected with water, or cRNA(s) for NHERF2, WNK4, or NHERF2 and WNK4 (12.5 ng/oocyte each), were injected with TRPV5 (12.5 ng/oocyte) 12 hours later. Ca 2+ uptake were performed 3 hours later (n =21). C . Assessment of stability of functional TRPV5 at the cell surface. Oocytes expressing TRPV5 (T5), TRPV5 and NHERF2 (T5+NF2), TRPV5 and WNK4 (T5+W4), and all the 3 proteins (T5+NF2+W4) for 36 hours were treated with Brefeldin A (BFA) at 20 μg/ml for indicated time period before Ca 2+ uptake experiments were performed. Ca 2+ uptake values (with the water injected control groups subtracted) are expressed as percentage of the value in the group with 0 hour BFA treatment (n=21). D . Plasma membrane distribution of FLAG-NHERF2 (green) and TRPV5 (red) as detected by co-immunostaining with anti-FLAG and anti-TRPV5 antibodies in oocytes co-expressing TRPV5 and FLAG-NHERF2. Bar, 50 μm.
    Figure Legend Snippet: NHERF2 did not alter the forward trafficking of TRPV5 but rendered TRPV5 more stable at the plasma membrane in the presence or absence of WNK4. A . NHERF2 didn’t improve the lack of robust response of N -glycosylation defective N358Q mutant of TRPV5 to the regulation by WNK4 (n = 14). B . Assessment of WNK4 and NHERF2 on TRPV5 forward trafficking. Oocytes injected with water, or cRNA(s) for NHERF2, WNK4, or NHERF2 and WNK4 (12.5 ng/oocyte each), were injected with TRPV5 (12.5 ng/oocyte) 12 hours later. Ca 2+ uptake were performed 3 hours later (n =21). C . Assessment of stability of functional TRPV5 at the cell surface. Oocytes expressing TRPV5 (T5), TRPV5 and NHERF2 (T5+NF2), TRPV5 and WNK4 (T5+W4), and all the 3 proteins (T5+NF2+W4) for 36 hours were treated with Brefeldin A (BFA) at 20 μg/ml for indicated time period before Ca 2+ uptake experiments were performed. Ca 2+ uptake values (with the water injected control groups subtracted) are expressed as percentage of the value in the group with 0 hour BFA treatment (n=21). D . Plasma membrane distribution of FLAG-NHERF2 (green) and TRPV5 (red) as detected by co-immunostaining with anti-FLAG and anti-TRPV5 antibodies in oocytes co-expressing TRPV5 and FLAG-NHERF2. Bar, 50 μm.

    Techniques Used: Mutagenesis, Injection, Functional Assay, Expressing, Immunostaining

    8) Product Images from "GADD34 function in protein trafficking promotes adaptation to hyperosmotic stress in human corneal cells"

    Article Title: GADD34 function in protein trafficking promotes adaptation to hyperosmotic stress in human corneal cells

    Journal: Cell reports

    doi: 10.1016/j.celrep.2017.11.027

    Hyperosmotic stress-induced GADD34/PP1 activity promotes post-ER SNAT2 protein processing in corneal epithelial cells A. Western blot analysis of extracts from cells treated with 500 mOsm media alone for 5h or supplemented with Sal003 (30 μM), Tunicamycin (Tu, 500 nM), Brefeldin A (BFA, 20 μM) or Golgicide A (GCA, 20 μM). Positions of protein size markers are indicated. B. Western blot analysis of total cell extracts from cells treated with 500 mOsm media for 5h with or without Sup. Sal003 (30 μM). MG132 (100 μM) was added for the last 1h of treatment. Cell extracts from cells treated with 500 mOsm media and Tunicamycin (Tu) or Brefeldin A (BFA) were analyzed by loading one-third the amount of the other samples. C. Quantification of immature (left) and unglycosylated (right) SNAT2 levels from cells treated as in panel B. Signal intensities were normalized to α-tubulin. D. Subcellular distribution of SNAT2 (green channel) and ER-resident proteins (red channel, visualized by anti-KDEL antibody staining) in cells grown in control or 500 mOsm media for 5h with or without Sal003 (30 μM) addition. Scale bars are 10 μm. E. Boxed image areas from panel D, white arrowheads indicate SNAT2 protein, dotted arrows point to KDEL-positive structures. Scale bar is 1 μm. F. Quantification of SNAT2 co-localization with KDEL reporter in cells exposed to 500 mOsm media with or without Sal003. Masks of SNAT2 and KDEL signals were created and overlap between areas was calculated in 4 separate planes. 9 and 11 cells were analyzed respectively. Data re represented as mean ± SD.
    Figure Legend Snippet: Hyperosmotic stress-induced GADD34/PP1 activity promotes post-ER SNAT2 protein processing in corneal epithelial cells A. Western blot analysis of extracts from cells treated with 500 mOsm media alone for 5h or supplemented with Sal003 (30 μM), Tunicamycin (Tu, 500 nM), Brefeldin A (BFA, 20 μM) or Golgicide A (GCA, 20 μM). Positions of protein size markers are indicated. B. Western blot analysis of total cell extracts from cells treated with 500 mOsm media for 5h with or without Sup. Sal003 (30 μM). MG132 (100 μM) was added for the last 1h of treatment. Cell extracts from cells treated with 500 mOsm media and Tunicamycin (Tu) or Brefeldin A (BFA) were analyzed by loading one-third the amount of the other samples. C. Quantification of immature (left) and unglycosylated (right) SNAT2 levels from cells treated as in panel B. Signal intensities were normalized to α-tubulin. D. Subcellular distribution of SNAT2 (green channel) and ER-resident proteins (red channel, visualized by anti-KDEL antibody staining) in cells grown in control or 500 mOsm media for 5h with or without Sal003 (30 μM) addition. Scale bars are 10 μm. E. Boxed image areas from panel D, white arrowheads indicate SNAT2 protein, dotted arrows point to KDEL-positive structures. Scale bar is 1 μm. F. Quantification of SNAT2 co-localization with KDEL reporter in cells exposed to 500 mOsm media with or without Sal003. Masks of SNAT2 and KDEL signals were created and overlap between areas was calculated in 4 separate planes. 9 and 11 cells were analyzed respectively. Data re represented as mean ± SD.

    Techniques Used: Activity Assay, Western Blot, Staining

    9) Product Images from "Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study"

    Article Title: Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13901

    Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P
    Figure Legend Snippet: Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P

    Techniques Used: Functional Assay, Inhibition

    Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).
    Figure Legend Snippet: Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).

    Techniques Used: Functional Assay, Blocking Assay

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    Article Snippet: .. Flow cytometry Following T cell culture, cells were resuspended in media containing phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Fisher Scientific), ionomycin (750 ng/mL, Fisher Scientific) and brefeldin A (5 ug/mL, Tocris Bioscience) for 4 hours. .. Cells were then fixed, permeabilized and stained with fluorescent-labelled antibodies for CD4, FoxP3, IL-17 and IL-22 using FoxP3 Fix/Perm Buffer Set (Biolegend).

    Cytometry:

    Article Title: Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors
    Article Snippet: .. Flow cytometry Following T cell culture, cells were resuspended in media containing phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Fisher Scientific), ionomycin (750 ng/mL, Fisher Scientific) and brefeldin A (5 ug/mL, Tocris Bioscience) for 4 hours. .. Cells were then fixed, permeabilized and stained with fluorescent-labelled antibodies for CD4, FoxP3, IL-17 and IL-22 using FoxP3 Fix/Perm Buffer Set (Biolegend).

    Cell Culture:

    Article Title: Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors
    Article Snippet: .. Flow cytometry Following T cell culture, cells were resuspended in media containing phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Fisher Scientific), ionomycin (750 ng/mL, Fisher Scientific) and brefeldin A (5 ug/mL, Tocris Bioscience) for 4 hours. .. Cells were then fixed, permeabilized and stained with fluorescent-labelled antibodies for CD4, FoxP3, IL-17 and IL-22 using FoxP3 Fix/Perm Buffer Set (Biolegend).

    Concentration Assay:

    Article Title: Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study
    Article Snippet: .. Stock solutions of brefeldin A and EEDQ were first prepared in pure DMSO and then diluted in aCSF to obtain a final concentration of 0.1% DMSO. .. It has been previously shown that 0.1% DMSO does not affect LC cell firing responses (Pineda et al. , ).

    Incubation:

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    Article Snippet: .. Then oocytes were incubated with Brefeldin A (Tocris Bioscience, Ellisville, MO) at 20 μg/ml for 0, 4, and 8 hours before Ca2+ uptake assay was carried out. .. Ca2+ uptake values were subtracted by those of oocytes injected with water and were expressed as percentage of the Ca2+ uptake value at 0 hour of incubation with BFA.

    other:

    Article Title: Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study
    Article Snippet: Brefeldin A is a macrocyclic transport inhibitor that has been used to slow down vacuolar protein trafficking in the endomembrane system of eukaryotic cells (Law et al. , ; Nebenführ et al. , ).

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    Article Title: Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study
    Article Snippet: Finally, when the slice temperature was lowered to 22°C or brefeldin A (10 μM) was perfused during the β‐FNA washout ( t 15 = 45), the recovery of ME effect was reduced by 46 ± 11% ( n = 5, P < 0.05 vs. control) and 66 ± 8% ( n = 5, P < 0.05 vs. control) respectively (Figure A, B).

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    Article Snippet: Cytochalasin D, nocodazole, brefeldin A, PF573228, vinblastine, and Y27632 were purchased from Tocris Chemical Co. (St. Louis, MO, USA).

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    Tocris brefeldin a
    CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of <t>brefeldin</t> A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).
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    CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).

    Journal: Scientific Reports

    Article Title: Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors

    doi: 10.1038/s41598-018-29262-4

    Figure Lengend Snippet: CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).

    Article Snippet: Flow cytometry Following T cell culture, cells were resuspended in media containing phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Fisher Scientific), ionomycin (750 ng/mL, Fisher Scientific) and brefeldin A (5 ug/mL, Tocris Bioscience) for 4 hours.

    Techniques: Purification, Selection, Cell Culture, Staining, Flow Cytometry, Cytometry

    Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P

    Journal: British Journal of Pharmacology

    Article Title: Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study

    doi: 10.1111/bph.13901

    Figure Lengend Snippet: Intracellular mechanisms of the functional turnover of μ receptors. (A) Bar histograms show the mean ± SEM of the percentage of ME‐induced effect immediately ( t 15 = 0) and 45 min after 15 min ( t 15 = 45) of β‐FNA (300 nM, 15 min) perfusion in different experimental conditions: control ( n = 6), in the presence of low‐calcium aCSF ( n = 6), during neuronal inhibition by NA (100 μM) ( n = 6), at low temperature ( n = 5) or during trafficking inhibition by brefeldin A (10 μM) ( n = 5). * P

    Article Snippet: Stock solutions of brefeldin A and EEDQ were first prepared in pure DMSO and then diluted in aCSF to obtain a final concentration of 0.1% DMSO.

    Techniques: Functional Assay, Inhibition

    Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).

    Journal: British Journal of Pharmacology

    Article Title: Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study) Characterization of functional μ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study

    doi: 10.1111/bph.13901

    Figure Lengend Snippet: Scheme summarizing the experimental design for functional characterization of μ receptor (MOR) turnover. (A) Functional turnover of μ receptor ‐mediated effect was evaluated by analysing the recovery of ME (3.2 μM, 1 min) effect after complete inactivation of μ receptors with the irreversible alkylating blocker β‐FNA (300–800 nM, 30 min); opioid effect recovery was measured before (control effect), immediately after inactivation ( t 30 = 0) and, then, every 15 min over a period of 300 min ( t 30 = 15–300). (B) For the μ receptor turnover to be compared with that of α 2 ‐adrenoceptors, NA (100 μM, 1 min) effect was tested before (control effect), immediately after complete receptor inactivation with the irreversible antagonist EEDQ (10 μM, 30 min) ( t 30 = 0) and, then, every 15 min over 300 min after inactivation ( t 30 = 15–300). (C) Functional μ receptor turnover was further characterized by evaluating the recovery of ME (3.2 μM, 1 min) effect for 120 min after complete μ receptor inactivation with 15, 30, 45 or 60 min of β‐FNA administration ( t 15, 30, 45, 60 = 0–120). (D) The recovery of ME (3.2 μM, 1 min) effect was also evaluated for 120 min after completion of a second 15 min perfusion with β‐FNA following the recovery of the first pre‐application ( t 15(1) = 0–120 and t 15(2) = 0–120). (E) The possible mechanisms underlying μ receptor turnover were explored by perfusion with a low‐calcium (0.2 mM) aCSF (to block LC cell calcium‐dependent mechanisms), administration of the α 2 ‐adrenoceptor agonist NA (100 μM) (to inhibit LC neuron firing‐dependent mechanisms), lowering the temperature of the chamber to 22°C (to evaluate vesicle movement mechanisms) or administration of the protein transport inhibitor brefeldin A (10 μM) (to test vesicle trafficking mechanisms); these manipulations were applied during the period of functional recovery after β‐FNA application, but regular conditions were restored 3–5 min before testing ME effect (45 min).

    Article Snippet: Stock solutions of brefeldin A and EEDQ were first prepared in pure DMSO and then diluted in aCSF to obtain a final concentration of 0.1% DMSO.

    Techniques: Functional Assay, Blocking Assay

    The effect of Epac inhibition on the DMR of different ligands. (a) DMR heatmap of AR ligands in the subclone A and in the 20 μM brefeldin A (BFA)- and RNAi Epac1-treated cells. The real responses of all ligands in the untreated cells were used for visualizing their DMR characteristics, while the treatment-induced net changes were used for better visualization of the effect of Epac inhibition. Only ligands that gave rise to a DMR of > 40 pm or an Epac inhibition-induced net change of > 40 pm were included in this analysis. (b–i) The real-time DMR of different ligands in cells without (control) or with brefeldin A- or Epac1 RNAi pretreatment: (b) epinephrine; (c) UK14,304; (d) isoproterenol; (e) Isoetharine; (f) cimaterol; (g) clenbuterol; (h) salbutamol; (i) betaxolol. All ligands were profiled at 10 μM. Data represents mean ± s.d. (n = 4).

    Journal: Scientific Reports

    Article Title: Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed ?2-Adrenergic Receptors

    doi: 10.1038/srep03828

    Figure Lengend Snippet: The effect of Epac inhibition on the DMR of different ligands. (a) DMR heatmap of AR ligands in the subclone A and in the 20 μM brefeldin A (BFA)- and RNAi Epac1-treated cells. The real responses of all ligands in the untreated cells were used for visualizing their DMR characteristics, while the treatment-induced net changes were used for better visualization of the effect of Epac inhibition. Only ligands that gave rise to a DMR of > 40 pm or an Epac inhibition-induced net change of > 40 pm were included in this analysis. (b–i) The real-time DMR of different ligands in cells without (control) or with brefeldin A- or Epac1 RNAi pretreatment: (b) epinephrine; (c) UK14,304; (d) isoproterenol; (e) Isoetharine; (f) cimaterol; (g) clenbuterol; (h) salbutamol; (i) betaxolol. All ligands were profiled at 10 μM. Data represents mean ± s.d. (n = 4).

    Article Snippet: Cytochalasin D, nocodazole, brefeldin A, PF573228, vinblastine, and Y27632 were purchased from Tocris Chemical Co. (St. Louis, MO, USA).

    Techniques: Inhibition