ibmx  (Tocris)

 
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  • 95
    Name:
    IBMX
    Description:
    PDE inhibitor non selective
    Catalog Number:
    2845
    Price:
    None
    Purity:
    ≥99% (HPLC)
    Category:
    Phosphodiesterase Inhibitors Phosphodiesterases Enzymes Pharmacology
    Formula:
    3,7-Dihydro-1-methyl-3-(2-methylpropyl)-1H-purine-2,6-dione
    Buy from Supplier


    Structured Review

    Tocris ibmx
    IBMX
    PDE inhibitor non selective
    https://www.bioz.com/result/ibmx/product/Tocris
    Average 95 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    ibmx - by Bioz Stars, 2020-09
    95/100 stars

    Images

    1) Product Images from "CRIS--A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending"

    Article Title: CRIS--A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003960

    CRIS is a novel target for cAMP. ( A ) Sequence comparison of CNBDs from different proteins. Sequence alignment of CNBDs from mCRIS, cyclic nucleotide-gated channels (bCNGA1, rCNGA4), a hyperpolarization activated and cyclic nucleotide-gated channel (mHCN2), a regulatory subunit from PKA (bPKARI-B), the exchange protein directly-activated by cAMP (hEPAC1), the bacterial catabolite activator protein (CAP), and the ELK1 channel from zebrafish (zELK). Amino acids that have been shown to be essential for ligand binding [1] are highlighted with asterisks. The β strand that functions as an intrinsic ligand in the ELK channels is highlighted in grey. Secondary structure elements are indicated below (β sheets: β 1–8, black arrows; α helices: αA–C, PBC, grey boxes). ( B ) M4T model of the presumed CNBD of mCRIS in the presence of cAMP. ( C ) Close-up view of the CNBD model of mCRIS indicating important interactions of side chain and backbone atoms with cAMP. ( D–G ) Analysis of cAMP binding using FRET. ( D ) Model demonstrating that binding of cAMP changes the conformation of the CNBD resulting in a change in FRET. ( E ) Representative traces for the change in cerulean (blue) and citrine (yellow) emission during perfusion of cit-mCNBD-cer expressing CHO cells with 3 mM 8-Br-cAMP. Arrow indicates start of perfusion. ( F ) Average change in FRET (normalized emission ratio cerulean/FRET-citrine) during perfusion of cit-mCNBD-cer expressing cells with 3 mM 8-Br-cAMP (CNBD 8-Br-cAMP), 40 µM NKH477/100 µM IBMX (CNBD NKH/IBMX), 3 mM 8-Br-cGMP (CNBD 8-Br-cGMP), and cit-mCNBD-R288Q-cer expressing cells with 3 mM 8-Br-cAMP (CNBD-RQ 8-Br-cAMP), and 40 µM NKH477/100 µM IBMX (CNBD-RQ NKH/IBMX). Arrow indicates start of perfusion. ( G ) Average change in FRET after 10 min of perfusion (mean ± s.d., black; 95% confident interval, dotted, grey). N numbers and p values are indicated.
    Figure Legend Snippet: CRIS is a novel target for cAMP. ( A ) Sequence comparison of CNBDs from different proteins. Sequence alignment of CNBDs from mCRIS, cyclic nucleotide-gated channels (bCNGA1, rCNGA4), a hyperpolarization activated and cyclic nucleotide-gated channel (mHCN2), a regulatory subunit from PKA (bPKARI-B), the exchange protein directly-activated by cAMP (hEPAC1), the bacterial catabolite activator protein (CAP), and the ELK1 channel from zebrafish (zELK). Amino acids that have been shown to be essential for ligand binding [1] are highlighted with asterisks. The β strand that functions as an intrinsic ligand in the ELK channels is highlighted in grey. Secondary structure elements are indicated below (β sheets: β 1–8, black arrows; α helices: αA–C, PBC, grey boxes). ( B ) M4T model of the presumed CNBD of mCRIS in the presence of cAMP. ( C ) Close-up view of the CNBD model of mCRIS indicating important interactions of side chain and backbone atoms with cAMP. ( D–G ) Analysis of cAMP binding using FRET. ( D ) Model demonstrating that binding of cAMP changes the conformation of the CNBD resulting in a change in FRET. ( E ) Representative traces for the change in cerulean (blue) and citrine (yellow) emission during perfusion of cit-mCNBD-cer expressing CHO cells with 3 mM 8-Br-cAMP. Arrow indicates start of perfusion. ( F ) Average change in FRET (normalized emission ratio cerulean/FRET-citrine) during perfusion of cit-mCNBD-cer expressing cells with 3 mM 8-Br-cAMP (CNBD 8-Br-cAMP), 40 µM NKH477/100 µM IBMX (CNBD NKH/IBMX), 3 mM 8-Br-cGMP (CNBD 8-Br-cGMP), and cit-mCNBD-R288Q-cer expressing cells with 3 mM 8-Br-cAMP (CNBD-RQ 8-Br-cAMP), and 40 µM NKH477/100 µM IBMX (CNBD-RQ NKH/IBMX). Arrow indicates start of perfusion. ( G ) Average change in FRET after 10 min of perfusion (mean ± s.d., black; 95% confident interval, dotted, grey). N numbers and p values are indicated.

    Techniques Used: Sequencing, Ligand Binding Assay, Binding Assay, Expressing

    2) Product Images from "Dopaminergic modulation of ganglion-cell photoreceptors"

    Article Title: Dopaminergic modulation of ganglion-cell photoreceptors

    Journal: The European Journal of Neuroscience

    doi: 10.1111/j.1460-9568.2011.07975.x

    Elevating intracellular cAMP levels by application of forskolin and IBMX mimics the action of dopamine on the ipRGC photocurrent
    Figure Legend Snippet: Elevating intracellular cAMP levels by application of forskolin and IBMX mimics the action of dopamine on the ipRGC photocurrent

    Techniques Used:

    3) Product Images from "Phosphatases control PKA-dependent functional microdomains at the outer mitochondrial membrane"

    Article Title: Phosphatases control PKA-dependent functional microdomains at the outer mitochondrial membrane

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1806318115

    Iso induces distinct PKA-dependent patterns in the cytosol and OMM without measurable differences in cAMP levels. ( A ) Coculture of cells expressing H187 or OMM-H187 subjected to increasing doses of Fsk. cAMP increases in response to Fsk were not different in the two compartments. ( Inset ) Average ± SEM of 21 H187-expressing cells and 12 OMM-H187-expressing cells in three independent experiments. F/I, Fsk 20 µM combined to IBMX 100 µM; ns, not significant. ( B ) Coculture of cells expressing AKAR4 or OMM-AKAR4 subjected to increasing doses of Iso. AKAR4 signals in response to Iso were significantly higher at the OMM than in the cytosol. ( Inset ) Average ± SEM of 23 AKAR4-expressing cells and eight OMM-AKAR4–expressing cells in three independent experiments. *** P
    Figure Legend Snippet: Iso induces distinct PKA-dependent patterns in the cytosol and OMM without measurable differences in cAMP levels. ( A ) Coculture of cells expressing H187 or OMM-H187 subjected to increasing doses of Fsk. cAMP increases in response to Fsk were not different in the two compartments. ( Inset ) Average ± SEM of 21 H187-expressing cells and 12 OMM-H187-expressing cells in three independent experiments. F/I, Fsk 20 µM combined to IBMX 100 µM; ns, not significant. ( B ) Coculture of cells expressing AKAR4 or OMM-AKAR4 subjected to increasing doses of Iso. AKAR4 signals in response to Iso were significantly higher at the OMM than in the cytosol. ( Inset ) Average ± SEM of 23 AKAR4-expressing cells and eight OMM-AKAR4–expressing cells in three independent experiments. *** P

    Techniques Used: Expressing

    Phosphatases are responsible for the different AKAR4 responses between the cytosol and OMM in NRVMs. ( A ) Western blotting of PKA components in soluble (S) and mitochondrial (M) fractions from three independent primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed purity of mitochondria and cytosol, respectively. ( B ) PKA-dependent phosphorylation kinetics measured by OMM-AKAR4 (red trace) or cytosolic AKAR4 (black trace) in NRVMs. Challenge with 20 µM Fsk and 100 µM IBMX (F/I) resulted in saturation of both sensors. Upon rinsing the stimuli, the termination kinetics of the two sensors (depending on phosphatases) was drastically different, with OMM-AKAR4 being the slower of the two. Shown is an experiment representative of at least three independent repeats. ( C ) Western blotting testing the presence of PP2B, PP2A, and PP1 in soluble and mitochondrial fractions from primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed the purity of mitochondria and cytosol, respectively. Shown is an experiment representative of three independent experiments. ( D ) Coculture of NRVMs expressing AKAR4 or OMM-AKAR4 challenged with Fsk (20 nM) followed by CalA (50 nM) and CsA (200 nM) to block phosphatases. Data shown are the average ± SEM of 39 AKAR4-expressing cells and 21 OMM-AKAR4-expressing cells in six independent experiments. *** P
    Figure Legend Snippet: Phosphatases are responsible for the different AKAR4 responses between the cytosol and OMM in NRVMs. ( A ) Western blotting of PKA components in soluble (S) and mitochondrial (M) fractions from three independent primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed purity of mitochondria and cytosol, respectively. ( B ) PKA-dependent phosphorylation kinetics measured by OMM-AKAR4 (red trace) or cytosolic AKAR4 (black trace) in NRVMs. Challenge with 20 µM Fsk and 100 µM IBMX (F/I) resulted in saturation of both sensors. Upon rinsing the stimuli, the termination kinetics of the two sensors (depending on phosphatases) was drastically different, with OMM-AKAR4 being the slower of the two. Shown is an experiment representative of at least three independent repeats. ( C ) Western blotting testing the presence of PP2B, PP2A, and PP1 in soluble and mitochondrial fractions from primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed the purity of mitochondria and cytosol, respectively. Shown is an experiment representative of three independent experiments. ( D ) Coculture of NRVMs expressing AKAR4 or OMM-AKAR4 challenged with Fsk (20 nM) followed by CalA (50 nM) and CsA (200 nM) to block phosphatases. Data shown are the average ± SEM of 39 AKAR4-expressing cells and 21 OMM-AKAR4-expressing cells in six independent experiments. *** P

    Techniques Used: Western Blot, Expressing, Blocking Assay

    4) Product Images from "MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish"

    Article Title: MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

    Journal: Chemistry & Biology

    doi: 10.1016/j.chembiol.2015.08.010

    MEKi Reverse Behaviors Caused by PDE4 Blockade/AC Activation (A) Schematic overview of small-molecule screen for suppressors of thigmotaxic behavior. (B and C) Images (B) and quantification (C) of behavior of zebrafish larvae in an open arena. Orange dots: thigmotaxic behavior; blue dots: non-thigmotaxic behavior. Experimental repetitions: n = 6/7 with 25–35 larvae per treatment condition. (D) Images of individual zebrafish larval traces in 96-well plate. Zebrafish were treated for 1 hr with rolipram followed by 1 hr treatment with or without MEKi, and tracked for 1 hr. (E) Histograms showing distribution of total distance moved (normalized values) in response to treatment conditions: DMSO (n = 143 larvae), rolipram (n = 185 larvae), rolipram with MEKi PD0325901 (n = 227 larvae), and MEKi PD0325901 (n = 149 larvae). (F) Total distance moved (normalized values from Figure 2 E). Means and bootstrapped 95% CIs are shown. Dotted lines indicate the upper and lower CI limits for the DMSO-treated fish to facilitate comparison with this group. Due to the non-normal distribution and unequal variance of data across groups, means were compared by assessing the bootstrapped 95% CI of the differences between groups. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM, MEKi 1.5 μM. ∗ p
    Figure Legend Snippet: MEKi Reverse Behaviors Caused by PDE4 Blockade/AC Activation (A) Schematic overview of small-molecule screen for suppressors of thigmotaxic behavior. (B and C) Images (B) and quantification (C) of behavior of zebrafish larvae in an open arena. Orange dots: thigmotaxic behavior; blue dots: non-thigmotaxic behavior. Experimental repetitions: n = 6/7 with 25–35 larvae per treatment condition. (D) Images of individual zebrafish larval traces in 96-well plate. Zebrafish were treated for 1 hr with rolipram followed by 1 hr treatment with or without MEKi, and tracked for 1 hr. (E) Histograms showing distribution of total distance moved (normalized values) in response to treatment conditions: DMSO (n = 143 larvae), rolipram (n = 185 larvae), rolipram with MEKi PD0325901 (n = 227 larvae), and MEKi PD0325901 (n = 149 larvae). (F) Total distance moved (normalized values from Figure 2 E). Means and bootstrapped 95% CIs are shown. Dotted lines indicate the upper and lower CI limits for the DMSO-treated fish to facilitate comparison with this group. Due to the non-normal distribution and unequal variance of data across groups, means were compared by assessing the bootstrapped 95% CI of the differences between groups. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM, MEKi 1.5 μM. ∗ p

    Techniques Used: Activation Assay, Fluorescence In Situ Hybridization

    PDE Blockade/AC Activation Stimulates Thigmotaxis and Hyperactivity in Zebrafish Larvae (A and B) Images and schematic representation of thigmotaxis in an open arena following PDE4 blockade/AC activation. (C and D) Quantification (C) and representative image (D) of zebrafish larvae (3 dpf) in 10-cm Petri dish. Larvae exhibiting thigmotaxis are highlighted with orange dots, and non-thigmotaxic larvae with blue dots. Experimental repetitions: n = 7 with 24–35 larvae per treatment condition. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM. (E) Thigmotaxic response following caffeine treatment. Experimental repetitions: n = 3 (in light and dark) with 35–43 larvae per treatment. (F) Images of individual zebrafish larval traces (1 hr of tracking) in 96-well plate wells. (G) Swimming activity of zebrafish larvae (5 dpf) treated with PDE4 blockers/cAMP activators in light or dark conditions (1 hr treatment, 30 min tracking in the light or dark). Experimental repetitions: n = 4 with 24 larvae per treatment condition. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM. (H) Histograms showing distribution of total distance moved (normalized values) in response to DMSO (larvae, n = 48) and rolipram (larvae, n = 48), with swimming activity followed over 5 hr. The last 10 min of the first and fifth hour are plotted. Dashed line indicates the approximate separation between the two populations. L, light conditions; D, dark conditions. ∗ p
    Figure Legend Snippet: PDE Blockade/AC Activation Stimulates Thigmotaxis and Hyperactivity in Zebrafish Larvae (A and B) Images and schematic representation of thigmotaxis in an open arena following PDE4 blockade/AC activation. (C and D) Quantification (C) and representative image (D) of zebrafish larvae (3 dpf) in 10-cm Petri dish. Larvae exhibiting thigmotaxis are highlighted with orange dots, and non-thigmotaxic larvae with blue dots. Experimental repetitions: n = 7 with 24–35 larvae per treatment condition. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM. (E) Thigmotaxic response following caffeine treatment. Experimental repetitions: n = 3 (in light and dark) with 35–43 larvae per treatment. (F) Images of individual zebrafish larval traces (1 hr of tracking) in 96-well plate wells. (G) Swimming activity of zebrafish larvae (5 dpf) treated with PDE4 blockers/cAMP activators in light or dark conditions (1 hr treatment, 30 min tracking in the light or dark). Experimental repetitions: n = 4 with 24 larvae per treatment condition. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM. (H) Histograms showing distribution of total distance moved (normalized values) in response to DMSO (larvae, n = 48) and rolipram (larvae, n = 48), with swimming activity followed over 5 hr. The last 10 min of the first and fifth hour are plotted. Dashed line indicates the approximate separation between the two populations. L, light conditions; D, dark conditions. ∗ p

    Techniques Used: Activation Assay, Activity Assay

    5) Product Images from "Phosphatases control PKA-dependent functional microdomains at the outer mitochondrial membrane"

    Article Title: Phosphatases control PKA-dependent functional microdomains at the outer mitochondrial membrane

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1806318115

    Iso induces distinct PKA-dependent patterns in the cytosol and OMM without measurable differences in cAMP levels. ( A ) Coculture of cells expressing H187 or OMM-H187 subjected to increasing doses of Fsk. cAMP increases in response to Fsk were not different in the two compartments. ( Inset ) Average ± SEM of 21 H187-expressing cells and 12 OMM-H187-expressing cells in three independent experiments. F/I, Fsk 20 µM combined to IBMX 100 µM; ns, not significant. ( B ) Coculture of cells expressing AKAR4 or OMM-AKAR4 subjected to increasing doses of Iso. AKAR4 signals in response to Iso were significantly higher at the OMM than in the cytosol. ( Inset ) Average ± SEM of 23 AKAR4-expressing cells and eight OMM-AKAR4–expressing cells in three independent experiments. *** P
    Figure Legend Snippet: Iso induces distinct PKA-dependent patterns in the cytosol and OMM without measurable differences in cAMP levels. ( A ) Coculture of cells expressing H187 or OMM-H187 subjected to increasing doses of Fsk. cAMP increases in response to Fsk were not different in the two compartments. ( Inset ) Average ± SEM of 21 H187-expressing cells and 12 OMM-H187-expressing cells in three independent experiments. F/I, Fsk 20 µM combined to IBMX 100 µM; ns, not significant. ( B ) Coculture of cells expressing AKAR4 or OMM-AKAR4 subjected to increasing doses of Iso. AKAR4 signals in response to Iso were significantly higher at the OMM than in the cytosol. ( Inset ) Average ± SEM of 23 AKAR4-expressing cells and eight OMM-AKAR4–expressing cells in three independent experiments. *** P

    Techniques Used: Expressing

    Phosphatases are responsible for the different AKAR4 responses between the cytosol and OMM in NRVMs. ( A ) Western blotting of PKA components in soluble (S) and mitochondrial (M) fractions from three independent primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed purity of mitochondria and cytosol, respectively. ( B ) PKA-dependent phosphorylation kinetics measured by OMM-AKAR4 (red trace) or cytosolic AKAR4 (black trace) in NRVMs. Challenge with 20 µM Fsk and 100 µM IBMX (F/I) resulted in saturation of both sensors. Upon rinsing the stimuli, the termination kinetics of the two sensors (depending on phosphatases) was drastically different, with OMM-AKAR4 being the slower of the two. Shown is an experiment representative of at least three independent repeats. ( C ) Western blotting testing the presence of PP2B, PP2A, and PP1 in soluble and mitochondrial fractions from primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed the purity of mitochondria and cytosol, respectively. Shown is an experiment representative of three independent experiments. ( D ) Coculture of NRVMs expressing AKAR4 or OMM-AKAR4 challenged with Fsk (20 nM) followed by CalA (50 nM) and CsA (200 nM) to block phosphatases. Data shown are the average ± SEM of 39 AKAR4-expressing cells and 21 OMM-AKAR4-expressing cells in six independent experiments. *** P
    Figure Legend Snippet: Phosphatases are responsible for the different AKAR4 responses between the cytosol and OMM in NRVMs. ( A ) Western blotting of PKA components in soluble (S) and mitochondrial (M) fractions from three independent primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed purity of mitochondria and cytosol, respectively. ( B ) PKA-dependent phosphorylation kinetics measured by OMM-AKAR4 (red trace) or cytosolic AKAR4 (black trace) in NRVMs. Challenge with 20 µM Fsk and 100 µM IBMX (F/I) resulted in saturation of both sensors. Upon rinsing the stimuli, the termination kinetics of the two sensors (depending on phosphatases) was drastically different, with OMM-AKAR4 being the slower of the two. Shown is an experiment representative of at least three independent repeats. ( C ) Western blotting testing the presence of PP2B, PP2A, and PP1 in soluble and mitochondrial fractions from primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed the purity of mitochondria and cytosol, respectively. Shown is an experiment representative of three independent experiments. ( D ) Coculture of NRVMs expressing AKAR4 or OMM-AKAR4 challenged with Fsk (20 nM) followed by CalA (50 nM) and CsA (200 nM) to block phosphatases. Data shown are the average ± SEM of 39 AKAR4-expressing cells and 21 OMM-AKAR4-expressing cells in six independent experiments. *** P

    Techniques Used: Western Blot, Expressing, Blocking Assay

    6) Product Images from "HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation"

    Article Title: HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation

    Journal: Science signaling

    doi: 10.1126/scisignal.aaa2581

    HDL-S1P acts as a biased agonist on S1P 1 ( A ) HUVECs were transduced with lentivirus encoding GFP-tagged S1P 1 and after serum starvation, were stimulated with TNFα or with human HDL (huHDL), albumin- S1P, or P-FTY720 for the indicated times. Confocal microscopy was performed to assess the localization of GFP-tagged S1P 1 . Images are representative of three independent experiments. ( B ) Serum-starved HUVECs were stimulated with albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylation was assessed by immunoblot of total lysates. ( C ) Serum-starved HUVECs were preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), and then stimulated with increasing doses of albumin-S1P, HDL-S1P, or ApoM − HDL in the presence of forskolin for 30 min. Results are expressed as picomole of cAMP per microgram of protein. Data represent combined analysis of five separate experiments and are expressed as means ± SD. ** P
    Figure Legend Snippet: HDL-S1P acts as a biased agonist on S1P 1 ( A ) HUVECs were transduced with lentivirus encoding GFP-tagged S1P 1 and after serum starvation, were stimulated with TNFα or with human HDL (huHDL), albumin- S1P, or P-FTY720 for the indicated times. Confocal microscopy was performed to assess the localization of GFP-tagged S1P 1 . Images are representative of three independent experiments. ( B ) Serum-starved HUVECs were stimulated with albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylation was assessed by immunoblot of total lysates. ( C ) Serum-starved HUVECs were preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), and then stimulated with increasing doses of albumin-S1P, HDL-S1P, or ApoM − HDL in the presence of forskolin for 30 min. Results are expressed as picomole of cAMP per microgram of protein. Data represent combined analysis of five separate experiments and are expressed as means ± SD. ** P

    Techniques Used: Transduction, Confocal Microscopy

    HDL-S1P acts as a biased agonist on S1P 1 ( A ) HUVECs were transduced with lentivirus encoding GFP-tagged S1P 1 and after serum starvation, were stimulated with TNFα or with human HDL (huHDL), albumin- S1P, or P-FTY720 for the indicated times. Confocal microscopy was performed to assess the localization of GFP-tagged S1P 1 . Images are representative of three independent experiments. ( B ) Serum-starved HUVECs were stimulated with albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylation was assessed by immunoblot of total lysates. ( C ) Serum-starved HUVECs were preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), and then stimulated with increasing doses of albumin-S1P, HDL-S1P, or ApoM − HDL in the presence of forskolin for 30 min. Results are expressed as picomole of cAMP per microgram of protein. Data represent combined analysis of five separate experiments and are expressed as means ± SD. ** P
    Figure Legend Snippet: HDL-S1P acts as a biased agonist on S1P 1 ( A ) HUVECs were transduced with lentivirus encoding GFP-tagged S1P 1 and after serum starvation, were stimulated with TNFα or with human HDL (huHDL), albumin- S1P, or P-FTY720 for the indicated times. Confocal microscopy was performed to assess the localization of GFP-tagged S1P 1 . Images are representative of three independent experiments. ( B ) Serum-starved HUVECs were stimulated with albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylation was assessed by immunoblot of total lysates. ( C ) Serum-starved HUVECs were preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), and then stimulated with increasing doses of albumin-S1P, HDL-S1P, or ApoM − HDL in the presence of forskolin for 30 min. Results are expressed as picomole of cAMP per microgram of protein. Data represent combined analysis of five separate experiments and are expressed as means ± SD. ** P

    Techniques Used: Transduction, Confocal Microscopy

    7) Product Images from "A novel biosensor to study cAMP dynamics in cilia and flagella"

    Article Title: A novel biosensor to study cAMP dynamics in cilia and flagella

    Journal: eLife

    doi: 10.7554/eLife.14052

    Characterisation of mlCNBD-FRET in HEK293 cells. ( A ) Calibration of mlCNBD-FRET in HEK293 cells. FRET was measured in a cuvette in a spectrofluorometer under basal conditions (ES), after permeabilization with 20 μM digitonin, and the following addition of increasing cAMP concentrations (in nM). According to the null-point calibration method, the difference in FRET ratio of the treated samples to the basal condition was determined and used to determine the basal cAMP concentration. ( B ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with 40 μM NKH477/500 μM IBMX (black). FRET has been measured using spectrofluorometer. Data are presented as mean ± S.D. DMSO (0.13%, red) has been used as a control; n = 3 for each condition. ( C ) Similar to part ( B ) after stimulation with 2 μM isoproterenol (black). ( D ) Similar to part ( B ) for stimulation with 3 mM SNP (black). ( E ) Change in the cerulean fluorescence lifetime measured using Fluorescence Lifetime Spectroscopy (FLS). Cells have been permeabilized with 20 μM digitonin followed by addition of cAMP. The cerulean fluorescence decay was recorded and fitted with a bi-exponential decay to calculate the lifetime. The mean of the two lifetimes was calculated and averaged over n = 3 experiments. ( F ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with different concentrations of NKH477. The FRET ratio has been calculated when reaching a maximum and normalized to the baseline ratio. Data is shown as mean ± S.D.; n = 4. ( G ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after alternatingly stimulating with 500 nM isoproterenol (black) followed by a wash-out with ES. As a control, cells were stimulated with buffer only (red). Data is shown as mean ± S.D.; n = 3. DOI: http://dx.doi.org/10.7554/eLife.14052.006
    Figure Legend Snippet: Characterisation of mlCNBD-FRET in HEK293 cells. ( A ) Calibration of mlCNBD-FRET in HEK293 cells. FRET was measured in a cuvette in a spectrofluorometer under basal conditions (ES), after permeabilization with 20 μM digitonin, and the following addition of increasing cAMP concentrations (in nM). According to the null-point calibration method, the difference in FRET ratio of the treated samples to the basal condition was determined and used to determine the basal cAMP concentration. ( B ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with 40 μM NKH477/500 μM IBMX (black). FRET has been measured using spectrofluorometer. Data are presented as mean ± S.D. DMSO (0.13%, red) has been used as a control; n = 3 for each condition. ( C ) Similar to part ( B ) after stimulation with 2 μM isoproterenol (black). ( D ) Similar to part ( B ) for stimulation with 3 mM SNP (black). ( E ) Change in the cerulean fluorescence lifetime measured using Fluorescence Lifetime Spectroscopy (FLS). Cells have been permeabilized with 20 μM digitonin followed by addition of cAMP. The cerulean fluorescence decay was recorded and fitted with a bi-exponential decay to calculate the lifetime. The mean of the two lifetimes was calculated and averaged over n = 3 experiments. ( F ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with different concentrations of NKH477. The FRET ratio has been calculated when reaching a maximum and normalized to the baseline ratio. Data is shown as mean ± S.D.; n = 4. ( G ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after alternatingly stimulating with 500 nM isoproterenol (black) followed by a wash-out with ES. As a control, cells were stimulated with buffer only (red). Data is shown as mean ± S.D.; n = 3. DOI: http://dx.doi.org/10.7554/eLife.14052.006

    Techniques Used: Concentration Assay, Expressing, Fluorescence, Spectroscopy

    8) Product Images from "Synaptic plasticity through activation of GluA3-containing AMPA-receptors"

    Article Title: Synaptic plasticity through activation of GluA3-containing AMPA-receptors

    Journal: eLife

    doi: 10.7554/eLife.25462

    Controls in FRAP experiment. ( A ) Fast fluorescence recovery of cytoplasmic TdTomato signal in photobleached spines. Left: tdTomato signal of spines transfected with SEP-GluA1 + tdTomato in the absence (dark blue; n=8) and the presence (light blue; n=6) of forskolin and IBMX (RM-ANOVA, p=0.09). Right: tdTomato signal of spines transfected with SEP-GluA3 + tdTomato in the absence (dark red; n=7) and the presence (light red; n=7) of forskolin and IBMX (RM-ANOVA, p=0.3). ( B ) SEP signal in neighboring control spines were unaffected after photobleaching. SEP signals normalized to tdTomato signal of control non-bleached spines transfected with SEP-GluA1 (left: n=6; RM-ANOVA, p=0.3) or SEP-GluA3 (right: n=5; RM-ANOVA, p=0.06) in the absence (dark) and the presence (light) of forskolin and IBMX. Error bars indicate SEM.
    Figure Legend Snippet: Controls in FRAP experiment. ( A ) Fast fluorescence recovery of cytoplasmic TdTomato signal in photobleached spines. Left: tdTomato signal of spines transfected with SEP-GluA1 + tdTomato in the absence (dark blue; n=8) and the presence (light blue; n=6) of forskolin and IBMX (RM-ANOVA, p=0.09). Right: tdTomato signal of spines transfected with SEP-GluA3 + tdTomato in the absence (dark red; n=7) and the presence (light red; n=7) of forskolin and IBMX (RM-ANOVA, p=0.3). ( B ) SEP signal in neighboring control spines were unaffected after photobleaching. SEP signals normalized to tdTomato signal of control non-bleached spines transfected with SEP-GluA1 (left: n=6; RM-ANOVA, p=0.3) or SEP-GluA3 (right: n=5; RM-ANOVA, p=0.06) in the absence (dark) and the presence (light) of forskolin and IBMX. Error bars indicate SEM.

    Techniques Used: Fluorescence, Transfection

    9) Product Images from "Ovarian hormones influence corticotropin releasing factor receptor colocalization with delta opioid receptors in CA1 pyramidal cell dendrites"

    Article Title: Ovarian hormones influence corticotropin releasing factor receptor colocalization with delta opioid receptors in CA1 pyramidal cell dendrites

    Journal: Experimental neurology

    doi: 10.1016/j.expneurol.2011.04.012

    CRF-induced increases in intracellular cAMP levels are mediated by the CRF receptor and attenuated by DOR activation. The cAMP levels were quantified in IBMX-pretreated transfected and untransfected cells after forskolin (10 μM), SNC80 (10 nM),
    Figure Legend Snippet: CRF-induced increases in intracellular cAMP levels are mediated by the CRF receptor and attenuated by DOR activation. The cAMP levels were quantified in IBMX-pretreated transfected and untransfected cells after forskolin (10 μM), SNC80 (10 nM),

    Techniques Used: Activation Assay, Transfection

    10) Product Images from "Mind the gap (junction): cGMP induced by nitric oxide in cardiac myocytes originates from cardiac fibroblasts, et al. Mind the gap (junction): cGMP induced by nitric oxide in cardiac myocytes originates from cardiac fibroblasts"

    Article Title: Mind the gap (junction): cGMP induced by nitric oxide in cardiac myocytes originates from cardiac fibroblasts, et al. Mind the gap (junction): cGMP induced by nitric oxide in cardiac myocytes originates from cardiac fibroblasts

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.14835

    NO–GC‐induced cGMP signals in cardiac fibroblasts. (a) Stimulation of cardiac fibroblasts by the indicated NO–GC‐activating compounds increased cGMP. CNP was applied as positive control; all in the presence of IBMX. Data shown are the time courses as means ± SEM of fibroblasts from N = 5 mice. (b) Effect of PDE inhibitors added to GSNO stimulation (1 nM). Data shown are individual values (for each inhibitor, one pair corresponds to measurements with fibroblasts from one mouse) before and after application of PDE inhibitors. * P
    Figure Legend Snippet: NO–GC‐induced cGMP signals in cardiac fibroblasts. (a) Stimulation of cardiac fibroblasts by the indicated NO–GC‐activating compounds increased cGMP. CNP was applied as positive control; all in the presence of IBMX. Data shown are the time courses as means ± SEM of fibroblasts from N = 5 mice. (b) Effect of PDE inhibitors added to GSNO stimulation (1 nM). Data shown are individual values (for each inhibitor, one pair corresponds to measurements with fibroblasts from one mouse) before and after application of PDE inhibitors. * P

    Techniques Used: Positive Control, Mouse Assay

    NO‐induced cGMP of cardiac fibroblasts. (a) Primary cardiac fibroblasts were stimulated for 5 min with the indicated GSNO concentrations. Values are means ± SEM of fibroblasts from N = 5 mice. (b–d) Cardiac (b) and dermal (c) fibroblasts and aortic smooth muscle cells (d) were stimulated with GSNO and IBMX as indicated and formed cGMP was determined by RIA. Data shown are individual values obtained from cells of one mouse each, together with means. * P
    Figure Legend Snippet: NO‐induced cGMP of cardiac fibroblasts. (a) Primary cardiac fibroblasts were stimulated for 5 min with the indicated GSNO concentrations. Values are means ± SEM of fibroblasts from N = 5 mice. (b–d) Cardiac (b) and dermal (c) fibroblasts and aortic smooth muscle cells (d) were stimulated with GSNO and IBMX as indicated and formed cGMP was determined by RIA. Data shown are individual values obtained from cells of one mouse each, together with means. * P

    Techniques Used: Mouse Assay

    11) Product Images from "Glucagon Increases Beating Rate but Not Contractility in Rat Right Atrium. Comparison with Isoproterenol"

    Article Title: Glucagon Increases Beating Rate but Not Contractility in Rat Right Atrium. Comparison with Isoproterenol

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0132884

    (A) Concentration response curves for the positive chronotropic effects of glucagon in the absence (▲) and in the presence of the non selective PDE inhibitor IBMX (3 μmol/L, ●), the selective PDE3 inhibitor cilostamide (0.3 μmol/L, ■) or the selective PDE4 inhibitor rolipram (1 μmol/L, ▼) in the spontaneously beating rat right atria. (B) Concentration response curves for the positive chronotropic effects of isoproterenol in the absence (▲) and in the presence of the non selective PDE inhibitor IBMX (3 μmol/L, ●) in the spontaneously beating rat right atria. Results are expressed as increase in basal rate (beats min -1 ). Both IBMX and rolipram increased beating rate by 26 ± 3 beats min -1 (n = 8) and 36 ± 6 beats min -1 (n = 5) respectively. Cilostamide did not changed basal atrial rate. When the preparation was treated with either IBMX or rolipram the beating rate in the presence of each of these agents was taken as the basal beating rate. Each point represents the mean value ± s.e.m (vertical bars) of 4–6 experiments.
    Figure Legend Snippet: (A) Concentration response curves for the positive chronotropic effects of glucagon in the absence (▲) and in the presence of the non selective PDE inhibitor IBMX (3 μmol/L, ●), the selective PDE3 inhibitor cilostamide (0.3 μmol/L, ■) or the selective PDE4 inhibitor rolipram (1 μmol/L, ▼) in the spontaneously beating rat right atria. (B) Concentration response curves for the positive chronotropic effects of isoproterenol in the absence (▲) and in the presence of the non selective PDE inhibitor IBMX (3 μmol/L, ●) in the spontaneously beating rat right atria. Results are expressed as increase in basal rate (beats min -1 ). Both IBMX and rolipram increased beating rate by 26 ± 3 beats min -1 (n = 8) and 36 ± 6 beats min -1 (n = 5) respectively. Cilostamide did not changed basal atrial rate. When the preparation was treated with either IBMX or rolipram the beating rate in the presence of each of these agents was taken as the basal beating rate. Each point represents the mean value ± s.e.m (vertical bars) of 4–6 experiments.

    Techniques Used: Concentration Assay

    12) Product Images from "Resveratrol enhances the inotropic effect but inhibits the proarrhythmic effect of sympathomimetic agents in rat myocardium"

    Article Title: Resveratrol enhances the inotropic effect but inhibits the proarrhythmic effect of sympathomimetic agents in rat myocardium

    Journal: PeerJ

    doi: 10.7717/peerj.3113

    Diltiazem abolish the inotropic effect of IBMX in rat ventricular myocardium. (A) Representative trace showing the effect of IBMX (30 µM) in a rat right ventricular strip. As can be seen, IBMX enhances basal contractility, an effect nullified when 5 µM of the L-type Ca2 + current inhibitor, diltiazem, is added. (B) Effect of IBMX (30 µM) alone and combined with 5 µM diltiazem (IBMX + DILT) in rat right ventricular strips. Inotropic responses are expressed as percentage of basal contractility. Further details as in legend to Fig. 1 . Each bar represents the mean value ± SEM of 4 experiments.
    Figure Legend Snippet: Diltiazem abolish the inotropic effect of IBMX in rat ventricular myocardium. (A) Representative trace showing the effect of IBMX (30 µM) in a rat right ventricular strip. As can be seen, IBMX enhances basal contractility, an effect nullified when 5 µM of the L-type Ca2 + current inhibitor, diltiazem, is added. (B) Effect of IBMX (30 µM) alone and combined with 5 µM diltiazem (IBMX + DILT) in rat right ventricular strips. Inotropic responses are expressed as percentage of basal contractility. Further details as in legend to Fig. 1 . Each bar represents the mean value ± SEM of 4 experiments.

    Techniques Used: Stripping Membranes

    13) Product Images from "HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation"

    Article Title: HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation

    Journal: Science signaling

    doi: 10.1126/scisignal.aaa2581

    HDL-S1P acts as a biased agonist on S1P 1 ( A ) HUVECs were transduced with lentivirus encoding GFP-tagged S1P 1 and after serum starvation, were stimulated with TNFα or with human HDL (huHDL), albumin- S1P, or P-FTY720 for the indicated times. Confocal microscopy was performed to assess the localization of GFP-tagged S1P 1 . Images are representative of three independent experiments. ( B ) Serum-starved HUVECs were stimulated with albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylation was assessed by immunoblot of total lysates. ( C ) Serum-starved HUVECs were preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), and then stimulated with increasing doses of albumin-S1P, HDL-S1P, or ApoM − HDL in the presence of forskolin for 30 min. Results are expressed as picomole of cAMP per microgram of protein. Data represent combined analysis of five separate experiments and are expressed as means ± SD. ** P
    Figure Legend Snippet: HDL-S1P acts as a biased agonist on S1P 1 ( A ) HUVECs were transduced with lentivirus encoding GFP-tagged S1P 1 and after serum starvation, were stimulated with TNFα or with human HDL (huHDL), albumin- S1P, or P-FTY720 for the indicated times. Confocal microscopy was performed to assess the localization of GFP-tagged S1P 1 . Images are representative of three independent experiments. ( B ) Serum-starved HUVECs were stimulated with albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylation was assessed by immunoblot of total lysates. ( C ) Serum-starved HUVECs were preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), and then stimulated with increasing doses of albumin-S1P, HDL-S1P, or ApoM − HDL in the presence of forskolin for 30 min. Results are expressed as picomole of cAMP per microgram of protein. Data represent combined analysis of five separate experiments and are expressed as means ± SD. ** P

    Techniques Used: Transduction, Confocal Microscopy

    HDL-S1P acts as a biased agonist on S1P 1 ( A ) HUVECs were transduced with lentivirus encoding GFP-tagged S1P 1 and after serum starvation, were stimulated with TNFα or with human HDL (huHDL), albumin- S1P, or P-FTY720 for the indicated times. Confocal microscopy was performed to assess the localization of GFP-tagged S1P 1 . Images are representative of three independent experiments. ( B ) Serum-starved HUVECs were stimulated with albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylation was assessed by immunoblot of total lysates. ( C ) Serum-starved HUVECs were preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), and then stimulated with increasing doses of albumin-S1P, HDL-S1P, or ApoM − HDL in the presence of forskolin for 30 min. Results are expressed as picomole of cAMP per microgram of protein. Data represent combined analysis of five separate experiments and are expressed as means ± SD. ** P
    Figure Legend Snippet: HDL-S1P acts as a biased agonist on S1P 1 ( A ) HUVECs were transduced with lentivirus encoding GFP-tagged S1P 1 and after serum starvation, were stimulated with TNFα or with human HDL (huHDL), albumin- S1P, or P-FTY720 for the indicated times. Confocal microscopy was performed to assess the localization of GFP-tagged S1P 1 . Images are representative of three independent experiments. ( B ) Serum-starved HUVECs were stimulated with albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylation was assessed by immunoblot of total lysates. ( C ) Serum-starved HUVECs were preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), and then stimulated with increasing doses of albumin-S1P, HDL-S1P, or ApoM − HDL in the presence of forskolin for 30 min. Results are expressed as picomole of cAMP per microgram of protein. Data represent combined analysis of five separate experiments and are expressed as means ± SD. ** P

    Techniques Used: Transduction, Confocal Microscopy

    14) Product Images from "Protein kinase A activates the Hippo pathway to modulate cell proliferation and differentiation"

    Article Title: Protein kinase A activates the Hippo pathway to modulate cell proliferation and differentiation

    Journal: Genes & Development

    doi: 10.1101/gad.219402.113

    YAP/TAZ mediate the effect of cAMP in adipogenesis. ( A ) 3T3-L1 cells were treated with 10 μM forskolin or 100 μM IBMX for 1 h, or serum-starved 3T3-L1 cells were treated with 5 μM KT5720 for 1 h, and YAP phosphorylation and TAZ
    Figure Legend Snippet: YAP/TAZ mediate the effect of cAMP in adipogenesis. ( A ) 3T3-L1 cells were treated with 10 μM forskolin or 100 μM IBMX for 1 h, or serum-starved 3T3-L1 cells were treated with 5 μM KT5720 for 1 h, and YAP phosphorylation and TAZ

    Techniques Used:

    Related Articles

    Incubation:

    Article Title: Ovarian hormones influence corticotropin releasing factor receptor colocalization with delta opioid receptors in CA1 pyramidal cell dendrites
    Article Snippet: .. Cells then were incubated in 100 μl DMEM containing 0.5 mM IBMX ± 10 nM (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80; Tocris Bioscience, Ellisville, MO), a highly selective non-peptide DOR agonist ( ; ), for 30 min at 37°C. .. 100 μl DMEM containing 10 μM Forskolin (Tocris) or 100 nM CRF (Tocris) in 0.5 mM IBMX ± 10 nM SNC80 was added to each well and the incubation continued at 37°C for 15 min, at which time cells were washed in ice cold PBS, 100 μl lysis reagent was added to each well, and the plate was shaken on a microplate shaker for 10-15 min. From each sample, 50 μl from the cell culture plate was transferred into the appropriate well of the immunoassay microtiter plate. cAMP was measured using the manufacturer’s Enzyme Immuno Assay Kit Protocol (Cell Signaling Technology).

    Transferring:

    Article Title: Synaptic plasticity through activation of GluA3-containing AMPA-receptors
    Article Snippet: .. Where indicated, the following drugs were added to the perfusion solution: forskolin (50 μM; Sigma), IBMX (50 μM; Tocris), KT5720 (4 μM; Tocris), PKI (2 μM; Calbiochem), ESI05 (10 μM; Biolog); Salirasib (10 μM; Tocris); or inside the recording pipette: cAMP (100 μM; Sigma), N002 (100 μM; Biolog), 8-CPT (20 μM; Tocris). .. During evoked recordings, a cut was made between CA1 and CA3, and picrotoxin (50 μM) and 2-chloroadenosine (4 μM; Tocris) were added to the bath.

    Expressing:

    Article Title: CRIS--A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending
    Article Snippet: .. CHO cells expressing the FRET sensors were perfused with buffer (140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 , 5 mM HEPES) with or without 40 µM NKH477, 100 µM IBMX (Tocris), and 3 mM 8-Br-cAMP/cGMP (BioLog) 1 min after starting the recording. .. Fluorescence was detected through 470/24 nm and 535/30 nm bandpass filters.

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    Tocris ibmx
    CRIS is a novel target for cAMP. ( A ) Sequence comparison of CNBDs from different proteins. Sequence alignment of CNBDs from mCRIS, cyclic nucleotide-gated channels (bCNGA1, rCNGA4), a hyperpolarization activated and cyclic nucleotide-gated channel (mHCN2), a regulatory subunit from PKA (bPKARI-B), the exchange protein directly-activated by cAMP (hEPAC1), the bacterial catabolite activator protein (CAP), and the ELK1 channel from zebrafish (zELK). Amino acids that have been shown to be essential for ligand binding [1] are highlighted with asterisks. The β strand that functions as an intrinsic ligand in the ELK channels is highlighted in grey. Secondary structure elements are indicated below (β sheets: β 1–8, black arrows; α helices: αA–C, PBC, grey boxes). ( B ) M4T model of the presumed CNBD of mCRIS in the presence of cAMP. ( C ) Close-up view of the CNBD model of mCRIS indicating important interactions of side chain and backbone atoms with cAMP. ( D–G ) Analysis of cAMP binding using FRET. ( D ) Model demonstrating that binding of cAMP changes the conformation of the CNBD resulting in a change in FRET. ( E ) Representative traces for the change in cerulean (blue) and citrine (yellow) emission during perfusion of cit-mCNBD-cer expressing CHO cells with 3 mM 8-Br-cAMP. Arrow indicates start of perfusion. ( F ) Average change in FRET (normalized emission ratio cerulean/FRET-citrine) during perfusion of cit-mCNBD-cer expressing cells with 3 mM 8-Br-cAMP (CNBD 8-Br-cAMP), 40 µM <t>NKH477/100</t> µM <t>IBMX</t> (CNBD NKH/IBMX), 3 mM 8-Br-cGMP (CNBD 8-Br-cGMP), and cit-mCNBD-R288Q-cer expressing cells with 3 mM 8-Br-cAMP (CNBD-RQ 8-Br-cAMP), and 40 µM NKH477/100 µM IBMX (CNBD-RQ NKH/IBMX). Arrow indicates start of perfusion. ( G ) Average change in FRET after 10 min of perfusion (mean ± s.d., black; 95% confident interval, dotted, grey). N numbers and p values are indicated.
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    CRIS is a novel target for cAMP. ( A ) Sequence comparison of CNBDs from different proteins. Sequence alignment of CNBDs from mCRIS, cyclic nucleotide-gated channels (bCNGA1, rCNGA4), a hyperpolarization activated and cyclic nucleotide-gated channel (mHCN2), a regulatory subunit from PKA (bPKARI-B), the exchange protein directly-activated by cAMP (hEPAC1), the bacterial catabolite activator protein (CAP), and the ELK1 channel from zebrafish (zELK). Amino acids that have been shown to be essential for ligand binding [1] are highlighted with asterisks. The β strand that functions as an intrinsic ligand in the ELK channels is highlighted in grey. Secondary structure elements are indicated below (β sheets: β 1–8, black arrows; α helices: αA–C, PBC, grey boxes). ( B ) M4T model of the presumed CNBD of mCRIS in the presence of cAMP. ( C ) Close-up view of the CNBD model of mCRIS indicating important interactions of side chain and backbone atoms with cAMP. ( D–G ) Analysis of cAMP binding using FRET. ( D ) Model demonstrating that binding of cAMP changes the conformation of the CNBD resulting in a change in FRET. ( E ) Representative traces for the change in cerulean (blue) and citrine (yellow) emission during perfusion of cit-mCNBD-cer expressing CHO cells with 3 mM 8-Br-cAMP. Arrow indicates start of perfusion. ( F ) Average change in FRET (normalized emission ratio cerulean/FRET-citrine) during perfusion of cit-mCNBD-cer expressing cells with 3 mM 8-Br-cAMP (CNBD 8-Br-cAMP), 40 µM NKH477/100 µM IBMX (CNBD NKH/IBMX), 3 mM 8-Br-cGMP (CNBD 8-Br-cGMP), and cit-mCNBD-R288Q-cer expressing cells with 3 mM 8-Br-cAMP (CNBD-RQ 8-Br-cAMP), and 40 µM NKH477/100 µM IBMX (CNBD-RQ NKH/IBMX). Arrow indicates start of perfusion. ( G ) Average change in FRET after 10 min of perfusion (mean ± s.d., black; 95% confident interval, dotted, grey). N numbers and p values are indicated.

    Journal: PLoS Genetics

    Article Title: CRIS--A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

    doi: 10.1371/journal.pgen.1003960

    Figure Lengend Snippet: CRIS is a novel target for cAMP. ( A ) Sequence comparison of CNBDs from different proteins. Sequence alignment of CNBDs from mCRIS, cyclic nucleotide-gated channels (bCNGA1, rCNGA4), a hyperpolarization activated and cyclic nucleotide-gated channel (mHCN2), a regulatory subunit from PKA (bPKARI-B), the exchange protein directly-activated by cAMP (hEPAC1), the bacterial catabolite activator protein (CAP), and the ELK1 channel from zebrafish (zELK). Amino acids that have been shown to be essential for ligand binding [1] are highlighted with asterisks. The β strand that functions as an intrinsic ligand in the ELK channels is highlighted in grey. Secondary structure elements are indicated below (β sheets: β 1–8, black arrows; α helices: αA–C, PBC, grey boxes). ( B ) M4T model of the presumed CNBD of mCRIS in the presence of cAMP. ( C ) Close-up view of the CNBD model of mCRIS indicating important interactions of side chain and backbone atoms with cAMP. ( D–G ) Analysis of cAMP binding using FRET. ( D ) Model demonstrating that binding of cAMP changes the conformation of the CNBD resulting in a change in FRET. ( E ) Representative traces for the change in cerulean (blue) and citrine (yellow) emission during perfusion of cit-mCNBD-cer expressing CHO cells with 3 mM 8-Br-cAMP. Arrow indicates start of perfusion. ( F ) Average change in FRET (normalized emission ratio cerulean/FRET-citrine) during perfusion of cit-mCNBD-cer expressing cells with 3 mM 8-Br-cAMP (CNBD 8-Br-cAMP), 40 µM NKH477/100 µM IBMX (CNBD NKH/IBMX), 3 mM 8-Br-cGMP (CNBD 8-Br-cGMP), and cit-mCNBD-R288Q-cer expressing cells with 3 mM 8-Br-cAMP (CNBD-RQ 8-Br-cAMP), and 40 µM NKH477/100 µM IBMX (CNBD-RQ NKH/IBMX). Arrow indicates start of perfusion. ( G ) Average change in FRET after 10 min of perfusion (mean ± s.d., black; 95% confident interval, dotted, grey). N numbers and p values are indicated.

    Article Snippet: CHO cells expressing the FRET sensors were perfused with buffer (140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 , 5 mM HEPES) with or without 40 µM NKH477, 100 µM IBMX (Tocris), and 3 mM 8-Br-cAMP/cGMP (BioLog) 1 min after starting the recording.

    Techniques: Sequencing, Ligand Binding Assay, Binding Assay, Expressing

    Elevating intracellular cAMP levels by application of forskolin and IBMX mimics the action of dopamine on the ipRGC photocurrent

    Journal: The European Journal of Neuroscience

    Article Title: Dopaminergic modulation of ganglion-cell photoreceptors

    doi: 10.1111/j.1460-9568.2011.07975.x

    Figure Lengend Snippet: Elevating intracellular cAMP levels by application of forskolin and IBMX mimics the action of dopamine on the ipRGC photocurrent

    Article Snippet: DMSO (dimethyl sulfoxide) was included in the stocks of two bath-applied drugs, forskolin ([3R-(3a,4ab,5b,6b,6aa,10a,10ab,10ba)]-a-(acetyloxy)-3-e-thenyldodecahydro-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1H-naphtho[2,1-b]pyran-1-one; Tocris) and IBMX (isobutylmethylxanthine; Tocris).

    Techniques:

    Iso induces distinct PKA-dependent patterns in the cytosol and OMM without measurable differences in cAMP levels. ( A ) Coculture of cells expressing H187 or OMM-H187 subjected to increasing doses of Fsk. cAMP increases in response to Fsk were not different in the two compartments. ( Inset ) Average ± SEM of 21 H187-expressing cells and 12 OMM-H187-expressing cells in three independent experiments. F/I, Fsk 20 µM combined to IBMX 100 µM; ns, not significant. ( B ) Coculture of cells expressing AKAR4 or OMM-AKAR4 subjected to increasing doses of Iso. AKAR4 signals in response to Iso were significantly higher at the OMM than in the cytosol. ( Inset ) Average ± SEM of 23 AKAR4-expressing cells and eight OMM-AKAR4–expressing cells in three independent experiments. *** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Phosphatases control PKA-dependent functional microdomains at the outer mitochondrial membrane

    doi: 10.1073/pnas.1806318115

    Figure Lengend Snippet: Iso induces distinct PKA-dependent patterns in the cytosol and OMM without measurable differences in cAMP levels. ( A ) Coculture of cells expressing H187 or OMM-H187 subjected to increasing doses of Fsk. cAMP increases in response to Fsk were not different in the two compartments. ( Inset ) Average ± SEM of 21 H187-expressing cells and 12 OMM-H187-expressing cells in three independent experiments. F/I, Fsk 20 µM combined to IBMX 100 µM; ns, not significant. ( B ) Coculture of cells expressing AKAR4 or OMM-AKAR4 subjected to increasing doses of Iso. AKAR4 signals in response to Iso were significantly higher at the OMM than in the cytosol. ( Inset ) Average ± SEM of 23 AKAR4-expressing cells and eight OMM-AKAR4–expressing cells in three independent experiments. *** P

    Article Snippet: Fsk, IBMX, CalA, CsA, and H89 were purchased from Tocris Bioscience.

    Techniques: Expressing

    Phosphatases are responsible for the different AKAR4 responses between the cytosol and OMM in NRVMs. ( A ) Western blotting of PKA components in soluble (S) and mitochondrial (M) fractions from three independent primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed purity of mitochondria and cytosol, respectively. ( B ) PKA-dependent phosphorylation kinetics measured by OMM-AKAR4 (red trace) or cytosolic AKAR4 (black trace) in NRVMs. Challenge with 20 µM Fsk and 100 µM IBMX (F/I) resulted in saturation of both sensors. Upon rinsing the stimuli, the termination kinetics of the two sensors (depending on phosphatases) was drastically different, with OMM-AKAR4 being the slower of the two. Shown is an experiment representative of at least three independent repeats. ( C ) Western blotting testing the presence of PP2B, PP2A, and PP1 in soluble and mitochondrial fractions from primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed the purity of mitochondria and cytosol, respectively. Shown is an experiment representative of three independent experiments. ( D ) Coculture of NRVMs expressing AKAR4 or OMM-AKAR4 challenged with Fsk (20 nM) followed by CalA (50 nM) and CsA (200 nM) to block phosphatases. Data shown are the average ± SEM of 39 AKAR4-expressing cells and 21 OMM-AKAR4-expressing cells in six independent experiments. *** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Phosphatases control PKA-dependent functional microdomains at the outer mitochondrial membrane

    doi: 10.1073/pnas.1806318115

    Figure Lengend Snippet: Phosphatases are responsible for the different AKAR4 responses between the cytosol and OMM in NRVMs. ( A ) Western blotting of PKA components in soluble (S) and mitochondrial (M) fractions from three independent primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed purity of mitochondria and cytosol, respectively. ( B ) PKA-dependent phosphorylation kinetics measured by OMM-AKAR4 (red trace) or cytosolic AKAR4 (black trace) in NRVMs. Challenge with 20 µM Fsk and 100 µM IBMX (F/I) resulted in saturation of both sensors. Upon rinsing the stimuli, the termination kinetics of the two sensors (depending on phosphatases) was drastically different, with OMM-AKAR4 being the slower of the two. Shown is an experiment representative of at least three independent repeats. ( C ) Western blotting testing the presence of PP2B, PP2A, and PP1 in soluble and mitochondrial fractions from primary NRVM cultures. An antibody mixture against the rodent OXPHOS subunits and GAPDH assessed the purity of mitochondria and cytosol, respectively. Shown is an experiment representative of three independent experiments. ( D ) Coculture of NRVMs expressing AKAR4 or OMM-AKAR4 challenged with Fsk (20 nM) followed by CalA (50 nM) and CsA (200 nM) to block phosphatases. Data shown are the average ± SEM of 39 AKAR4-expressing cells and 21 OMM-AKAR4-expressing cells in six independent experiments. *** P

    Article Snippet: Fsk, IBMX, CalA, CsA, and H89 were purchased from Tocris Bioscience.

    Techniques: Western Blot, Expressing, Blocking Assay

    MEKi Reverse Behaviors Caused by PDE4 Blockade/AC Activation (A) Schematic overview of small-molecule screen for suppressors of thigmotaxic behavior. (B and C) Images (B) and quantification (C) of behavior of zebrafish larvae in an open arena. Orange dots: thigmotaxic behavior; blue dots: non-thigmotaxic behavior. Experimental repetitions: n = 6/7 with 25–35 larvae per treatment condition. (D) Images of individual zebrafish larval traces in 96-well plate. Zebrafish were treated for 1 hr with rolipram followed by 1 hr treatment with or without MEKi, and tracked for 1 hr. (E) Histograms showing distribution of total distance moved (normalized values) in response to treatment conditions: DMSO (n = 143 larvae), rolipram (n = 185 larvae), rolipram with MEKi PD0325901 (n = 227 larvae), and MEKi PD0325901 (n = 149 larvae). (F) Total distance moved (normalized values from Figure 2 E). Means and bootstrapped 95% CIs are shown. Dotted lines indicate the upper and lower CI limits for the DMSO-treated fish to facilitate comparison with this group. Due to the non-normal distribution and unequal variance of data across groups, means were compared by assessing the bootstrapped 95% CI of the differences between groups. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM, MEKi 1.5 μM. ∗ p

    Journal: Chemistry & Biology

    Article Title: MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

    doi: 10.1016/j.chembiol.2015.08.010

    Figure Lengend Snippet: MEKi Reverse Behaviors Caused by PDE4 Blockade/AC Activation (A) Schematic overview of small-molecule screen for suppressors of thigmotaxic behavior. (B and C) Images (B) and quantification (C) of behavior of zebrafish larvae in an open arena. Orange dots: thigmotaxic behavior; blue dots: non-thigmotaxic behavior. Experimental repetitions: n = 6/7 with 25–35 larvae per treatment condition. (D) Images of individual zebrafish larval traces in 96-well plate. Zebrafish were treated for 1 hr with rolipram followed by 1 hr treatment with or without MEKi, and tracked for 1 hr. (E) Histograms showing distribution of total distance moved (normalized values) in response to treatment conditions: DMSO (n = 143 larvae), rolipram (n = 185 larvae), rolipram with MEKi PD0325901 (n = 227 larvae), and MEKi PD0325901 (n = 149 larvae). (F) Total distance moved (normalized values from Figure 2 E). Means and bootstrapped 95% CIs are shown. Dotted lines indicate the upper and lower CI limits for the DMSO-treated fish to facilitate comparison with this group. Due to the non-normal distribution and unequal variance of data across groups, means were compared by assessing the bootstrapped 95% CI of the differences between groups. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM, MEKi 1.5 μM. ∗ p

    Article Snippet: Drug Treatments, Thigmotaxis Assay, and Small-Molecule Screen For zebrafish chemical treatments, forskolin, rolipram, and IBMX (all from Tocris Bioscience), caffeine (Sigma-Aldrich), and PD0325901 (University of Dundee, UK) were prepared in DMSO.

    Techniques: Activation Assay, Fluorescence In Situ Hybridization

    PDE Blockade/AC Activation Stimulates Thigmotaxis and Hyperactivity in Zebrafish Larvae (A and B) Images and schematic representation of thigmotaxis in an open arena following PDE4 blockade/AC activation. (C and D) Quantification (C) and representative image (D) of zebrafish larvae (3 dpf) in 10-cm Petri dish. Larvae exhibiting thigmotaxis are highlighted with orange dots, and non-thigmotaxic larvae with blue dots. Experimental repetitions: n = 7 with 24–35 larvae per treatment condition. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM. (E) Thigmotaxic response following caffeine treatment. Experimental repetitions: n = 3 (in light and dark) with 35–43 larvae per treatment. (F) Images of individual zebrafish larval traces (1 hr of tracking) in 96-well plate wells. (G) Swimming activity of zebrafish larvae (5 dpf) treated with PDE4 blockers/cAMP activators in light or dark conditions (1 hr treatment, 30 min tracking in the light or dark). Experimental repetitions: n = 4 with 24 larvae per treatment condition. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM. (H) Histograms showing distribution of total distance moved (normalized values) in response to DMSO (larvae, n = 48) and rolipram (larvae, n = 48), with swimming activity followed over 5 hr. The last 10 min of the first and fifth hour are plotted. Dashed line indicates the approximate separation between the two populations. L, light conditions; D, dark conditions. ∗ p

    Journal: Chemistry & Biology

    Article Title: MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

    doi: 10.1016/j.chembiol.2015.08.010

    Figure Lengend Snippet: PDE Blockade/AC Activation Stimulates Thigmotaxis and Hyperactivity in Zebrafish Larvae (A and B) Images and schematic representation of thigmotaxis in an open arena following PDE4 blockade/AC activation. (C and D) Quantification (C) and representative image (D) of zebrafish larvae (3 dpf) in 10-cm Petri dish. Larvae exhibiting thigmotaxis are highlighted with orange dots, and non-thigmotaxic larvae with blue dots. Experimental repetitions: n = 7 with 24–35 larvae per treatment condition. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM. (E) Thigmotaxic response following caffeine treatment. Experimental repetitions: n = 3 (in light and dark) with 35–43 larvae per treatment. (F) Images of individual zebrafish larval traces (1 hr of tracking) in 96-well plate wells. (G) Swimming activity of zebrafish larvae (5 dpf) treated with PDE4 blockers/cAMP activators in light or dark conditions (1 hr treatment, 30 min tracking in the light or dark). Experimental repetitions: n = 4 with 24 larvae per treatment condition. Drug treatment concentrations: rolipram 15 μM, forskolin 7.5 μM, IBMX 30 μM. (H) Histograms showing distribution of total distance moved (normalized values) in response to DMSO (larvae, n = 48) and rolipram (larvae, n = 48), with swimming activity followed over 5 hr. The last 10 min of the first and fifth hour are plotted. Dashed line indicates the approximate separation between the two populations. L, light conditions; D, dark conditions. ∗ p

    Article Snippet: Drug Treatments, Thigmotaxis Assay, and Small-Molecule Screen For zebrafish chemical treatments, forskolin, rolipram, and IBMX (all from Tocris Bioscience), caffeine (Sigma-Aldrich), and PD0325901 (University of Dundee, UK) were prepared in DMSO.

    Techniques: Activation Assay, Activity Assay