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  • 94
    Thermo Fisher ibmx
    Continuous exposure to albuterol reduces stimulated <t>CFTR</t> activity in a fashion similar to continuous or intermittent dosing with formoterol. wtCFTR + and VX809-corrected F508del-CFTR + CFBE41o- cells were exposed to either continuous or intermittent (1 hour, twice daily) albuterol (10 μM) or the long-acting β2AR-agonist formoterol (LABA; 1 μM) for 72 hours. Cells were mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. ( A ) Representative short-circuit current (I sc ) tracings in wtCFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in B ( n = 4 inserts/condition; circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]). ( C ) Representative I sc tracings in VX809-treated F508del-CFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in D ( n = 4 inserts/condition). All data were normalized to the control or VX809 (no albuterol pretreatment) condition. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (10 μM forskolin/100 μM <t>IBMX;</t> dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR + , 1 μM VX770 for F508del-CFTR + cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). All experiments are representative of studies repeated in duplicate or triplicate with similar results. Data presented represent the mean ± SEM. * P
    Ibmx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 182 article reviews
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    99
    Millipore ibmx
    Influence of a gradual decrease in the concentrations of insulin, dexamethasone, <t>rosiglitazone,</t> 3-isobutyl-1-methylxanthine <t>(IBMX)</t> and biotin in the induction medium to 30% and 10% of their original concentrations on lipid incorporation by bovine ASC. The ‘100% medium’ contained 10 µg/mL insulin, 1 µM dexamethasone, 20 µM rosiglitazone, 250 µM IBMX and 33 µM biotin in the recipes used by Riedel et al. [ 22 ]. The results are presented as means ± SEM of three independent experiments with two replicates. *Asterisks indicate an effect of factor day with P
    Ibmx, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3566 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ibmx/product/Millipore
    Average 99 stars, based on 3566 article reviews
    Price from $9.99 to $1999.99
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    ibmx  (Tocris)
    95
    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.
    Ibmx, supplied by Tocris, used in various techniques. Bioz Stars score: 95/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore isobutylmethylxanthine 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.
    Isobutylmethylxanthine Ibmx, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 400 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 400 article reviews
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    Image Search Results


    Continuous exposure to albuterol reduces stimulated CFTR activity in a fashion similar to continuous or intermittent dosing with formoterol. wtCFTR + and VX809-corrected F508del-CFTR + CFBE41o- cells were exposed to either continuous or intermittent (1 hour, twice daily) albuterol (10 μM) or the long-acting β2AR-agonist formoterol (LABA; 1 μM) for 72 hours. Cells were mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. ( A ) Representative short-circuit current (I sc ) tracings in wtCFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in B ( n = 4 inserts/condition; circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]). ( C ) Representative I sc tracings in VX809-treated F508del-CFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in D ( n = 4 inserts/condition). All data were normalized to the control or VX809 (no albuterol pretreatment) condition. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (10 μM forskolin/100 μM IBMX; dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR + , 1 μM VX770 for F508del-CFTR + cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). All experiments are representative of studies repeated in duplicate or triplicate with similar results. Data presented represent the mean ± SEM. * P

    Journal: JCI Insight

    Article Title: Chronic β2AR stimulation limits CFTR activation in human airway epithelia

    doi: 10.1172/jci.insight.93029

    Figure Lengend Snippet: Continuous exposure to albuterol reduces stimulated CFTR activity in a fashion similar to continuous or intermittent dosing with formoterol. wtCFTR + and VX809-corrected F508del-CFTR + CFBE41o- cells were exposed to either continuous or intermittent (1 hour, twice daily) albuterol (10 μM) or the long-acting β2AR-agonist formoterol (LABA; 1 μM) for 72 hours. Cells were mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. ( A ) Representative short-circuit current (I sc ) tracings in wtCFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in B ( n = 4 inserts/condition; circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]). ( C ) Representative I sc tracings in VX809-treated F508del-CFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in D ( n = 4 inserts/condition). All data were normalized to the control or VX809 (no albuterol pretreatment) condition. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (10 μM forskolin/100 μM IBMX; dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR + , 1 μM VX770 for F508del-CFTR + cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). All experiments are representative of studies repeated in duplicate or triplicate with similar results. Data presented represent the mean ± SEM. * P

    Article Snippet: CFTR was activated with apical/basolateral forskolin (10 μM; Sigma-Aldrich) and IBMX (100 μM; Acros Organics) and then potentiated with apical genistein (50 μM, Sigma-Aldrich, wtCFTR) or VX770 (1 μM, Selleck Chemicals, F508del/F508del).

    Techniques: Activity Assay, Inhibition

    Chronic albuterol-induced CFTR impairment is rescued with application of exogenous cAMP. wtCFTR + and VX809-corrected F508del-CFTR + CFBE41o- cells were exposed to albuterol and/or VX809 in media for 72 hours, then mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. In contrast to previously demonstrated experiments, cell-permeant 8-bromoadenosine cAMP (8-Br cAMP) was used to stimulate CFTR function to bypass cellular mechanisms of cAMP generation. In wtCFTR + cells (representative short-circuit current [I sc ] tracing in A , aggregate data in B ; n = 4 inserts/condition. Circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]), maximal stimulation with 8-Br cAMP in place of forskolin/IBMX rescues the previously demonstrated albuterol-induced CFTR dysfunction. Stimulation with increasing doses of 8-Br cAMP ( C , n = 3 inserts/data point) revealed a small increase in CFTR function in albuterol-pretreated cells at low stimulation doses. Squares, control conditions; triangles, albuterol pretreated. Similarly, in VX809-corrected F508del-CFTR + cells (representative I sc tracing in D , aggregate data in E ; n = 4 inserts/condition), maximal stimulation with 8-Br cAMP rescues albuterol-induced CFTR dysfunction. Unlike wtCFTR + cells, stimulation with increasing doses of 8-Br cAMP demonstrated no difference in VX809-corrected F508del-CFTR function following albuterol pretreatment at any stimulation dose ( F , n = 3 inserts/data point. All indicators of statistical significance are comparisons with untreated cells; no difference was noted between VX809-corrected cells with or without albuterol). Circles, untreated; squares, VX809 alone; triangles, VX809 + albuterol. All studies are internally normalized as indicated to allow for comparisons. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (100 μM 8-Br cAMP; dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR + ; 1 μM VX770 for F508del-CFTR + cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). Both experiments are representative of studies repeated in duplicate with similar results. Data presented represent the mean ± SEM. * P

    Journal: JCI Insight

    Article Title: Chronic β2AR stimulation limits CFTR activation in human airway epithelia

    doi: 10.1172/jci.insight.93029

    Figure Lengend Snippet: Chronic albuterol-induced CFTR impairment is rescued with application of exogenous cAMP. wtCFTR + and VX809-corrected F508del-CFTR + CFBE41o- cells were exposed to albuterol and/or VX809 in media for 72 hours, then mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. In contrast to previously demonstrated experiments, cell-permeant 8-bromoadenosine cAMP (8-Br cAMP) was used to stimulate CFTR function to bypass cellular mechanisms of cAMP generation. In wtCFTR + cells (representative short-circuit current [I sc ] tracing in A , aggregate data in B ; n = 4 inserts/condition. Circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]), maximal stimulation with 8-Br cAMP in place of forskolin/IBMX rescues the previously demonstrated albuterol-induced CFTR dysfunction. Stimulation with increasing doses of 8-Br cAMP ( C , n = 3 inserts/data point) revealed a small increase in CFTR function in albuterol-pretreated cells at low stimulation doses. Squares, control conditions; triangles, albuterol pretreated. Similarly, in VX809-corrected F508del-CFTR + cells (representative I sc tracing in D , aggregate data in E ; n = 4 inserts/condition), maximal stimulation with 8-Br cAMP rescues albuterol-induced CFTR dysfunction. Unlike wtCFTR + cells, stimulation with increasing doses of 8-Br cAMP demonstrated no difference in VX809-corrected F508del-CFTR function following albuterol pretreatment at any stimulation dose ( F , n = 3 inserts/data point. All indicators of statistical significance are comparisons with untreated cells; no difference was noted between VX809-corrected cells with or without albuterol). Circles, untreated; squares, VX809 alone; triangles, VX809 + albuterol. All studies are internally normalized as indicated to allow for comparisons. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (100 μM 8-Br cAMP; dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR + ; 1 μM VX770 for F508del-CFTR + cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). Both experiments are representative of studies repeated in duplicate with similar results. Data presented represent the mean ± SEM. * P

    Article Snippet: CFTR was activated with apical/basolateral forskolin (10 μM; Sigma-Aldrich) and IBMX (100 μM; Acros Organics) and then potentiated with apical genistein (50 μM, Sigma-Aldrich, wtCFTR) or VX770 (1 μM, Selleck Chemicals, F508del/F508del).

    Techniques: Activity Assay, Inhibition

    Chronic albuterol exposure reduces forskolin/IBMX–stimulated generation of cAMP in wtCFTR + and F508del-CFTR + CFBE41o- cells. wtCFTR + and F508del-CFTR + CFBE41o- cells were pretreated with albuterol (10 μM) for 72 hours, then stimulated to produce cAMP with 10 μM forskolin/100 μM IBMX (10 minutes). cAMP levels were then analyzed by colorimetric ELISA. wtCFTR + ( A , n = 6) and F508del-CFTR + ( B , n = 6) CFBE41o- cells produce minimal cAMP in the absence of either pretreatment or stimulation (open circles). Stimulation with 10 μM forskolin/100 μM IBMX in untreated cells leads to cAMP production (open squares), which is reduced by more than 60% in cells pretreated for 72 hours with albuterol (open triangles). All studies are internally normalized to control conditions to allow for comparisons. Both experiments are representative of studies repeated in duplicate with similar results. Data presented represent the mean ± SEM. n = 6 wells per condition. **** P

    Journal: JCI Insight

    Article Title: Chronic β2AR stimulation limits CFTR activation in human airway epithelia

    doi: 10.1172/jci.insight.93029

    Figure Lengend Snippet: Chronic albuterol exposure reduces forskolin/IBMX–stimulated generation of cAMP in wtCFTR + and F508del-CFTR + CFBE41o- cells. wtCFTR + and F508del-CFTR + CFBE41o- cells were pretreated with albuterol (10 μM) for 72 hours, then stimulated to produce cAMP with 10 μM forskolin/100 μM IBMX (10 minutes). cAMP levels were then analyzed by colorimetric ELISA. wtCFTR + ( A , n = 6) and F508del-CFTR + ( B , n = 6) CFBE41o- cells produce minimal cAMP in the absence of either pretreatment or stimulation (open circles). Stimulation with 10 μM forskolin/100 μM IBMX in untreated cells leads to cAMP production (open squares), which is reduced by more than 60% in cells pretreated for 72 hours with albuterol (open triangles). All studies are internally normalized to control conditions to allow for comparisons. Both experiments are representative of studies repeated in duplicate with similar results. Data presented represent the mean ± SEM. n = 6 wells per condition. **** P

    Article Snippet: CFTR was activated with apical/basolateral forskolin (10 μM; Sigma-Aldrich) and IBMX (100 μM; Acros Organics) and then potentiated with apical genistein (50 μM, Sigma-Aldrich, wtCFTR) or VX770 (1 μM, Selleck Chemicals, F508del/F508del).

    Techniques: Enzyme-linked Immunosorbent Assay

    Influence of a gradual decrease in the concentrations of insulin, dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine (IBMX) and biotin in the induction medium to 30% and 10% of their original concentrations on lipid incorporation by bovine ASC. The ‘100% medium’ contained 10 µg/mL insulin, 1 µM dexamethasone, 20 µM rosiglitazone, 250 µM IBMX and 33 µM biotin in the recipes used by Riedel et al. [ 22 ]. The results are presented as means ± SEM of three independent experiments with two replicates. *Asterisks indicate an effect of factor day with P

    Journal: Adipocyte

    Article Title: Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction

    doi: 10.1080/21623945.2020.1720480

    Figure Lengend Snippet: Influence of a gradual decrease in the concentrations of insulin, dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine (IBMX) and biotin in the induction medium to 30% and 10% of their original concentrations on lipid incorporation by bovine ASC. The ‘100% medium’ contained 10 µg/mL insulin, 1 µM dexamethasone, 20 µM rosiglitazone, 250 µM IBMX and 33 µM biotin in the recipes used by Riedel et al. [ 22 ]. The results are presented as means ± SEM of three independent experiments with two replicates. *Asterisks indicate an effect of factor day with P

    Article Snippet: N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), amphotericin B, AsA, biotin, pantothenic acid, bovine insulin, rosiglitazone, dexamethasone, IBMX, Nile red, TritonTM X-100, gelatin-A, poly-L-lysine, ß-glycerophosphate, 1,25-dihydroxyvitamin D3 , alizarin red (sodium sulphonate salt) and D-glucose were purchased from Sigma Aldrich (Taufkirchen, Germany).

    Techniques:

    LCoR inhibits the transcriptional activity of C/EBPβ and mitotic clonal expansion. A–C , HEK 293T cells were transiently transfected with a luciferase reporter plasmid containing PPAR γ 2 promoter (−602 to +52) ( PPAR γ 2-luc ) with or without a C/EBPβ construct in the absence or presence of increasing amounts of LCoR ( A ), siRNAs targeting LCoR ( B ), or full-length or truncated LCoR as indicated ( C ). The luciferase activity was measured and is shown as relative luciferase units ( RLU ). D–F , 3T3-L1 cells were infected with retrovirus expressing a PPAR γ 2 promoter (−602 to +52)-driven luciferase reporter with or without retrovirus expressing F-LCoR ( D ), shLCoR ( E ), or truncated LCoR ( F ) as indicated. The luciferase activities were analyzed before or 48 h after differentiation induction using insulin, dexamethasone, and 3-isobutyl-1-methylxanthine ( IDM ) and shown as relative luciferase units ( RLU ). G and H , representative image ( G ) and percentages of BrdU-positive cells ( H ) are shown. 18 h after adipogenic induction, 3T3-L1 cells stably overexpressing F-LCoR or an empty vector (pMSCV) were labeled with BrdU for 2 h and then stained with DAPI. The fluorescence of BrdU ( green ) and DAPI ( blue ) was detected with a fluorescence microscope. I–L , 3T3-L1 cells stably overexpressing F-LCoR or pMSCV were harvested at the indicated times after adipogenic induction. The mRNA levels of CCND2 ( I ), CCNE1 ( J ), Gins1 ( K ), and Mcm3 ( L ) were measured by qPCR analysis. M , the efficiency of the knockdown of C/EBPβ in 3T3-L1 cells was determined by Western blotting. 3T3-L1 preadipocytes were infected with a retroviral vector containing shC/EBPβ or shLacZ as a control. N–Q , 3T3-L1 cells infected with shC/EBPβ or shLacZ were harvested at the indicated times after adipogenic induction. The mRNA levels of CCND2 ( N ), CCNE1 ( O ), Gins1 ( P ), and Mcm3 ( Q ) were measured by qPCR analysis. Blots shown here are representative of at least three independent experiments. Data are represented as mean ± S.D. ( n = 3–4). Error bars represent S.D. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Ligand-dependent corepressor (LCoR) represses the transcription factor C/EBPβ during early adipocyte differentiation

    doi: 10.1074/jbc.M117.793984

    Figure Lengend Snippet: LCoR inhibits the transcriptional activity of C/EBPβ and mitotic clonal expansion. A–C , HEK 293T cells were transiently transfected with a luciferase reporter plasmid containing PPAR γ 2 promoter (−602 to +52) ( PPAR γ 2-luc ) with or without a C/EBPβ construct in the absence or presence of increasing amounts of LCoR ( A ), siRNAs targeting LCoR ( B ), or full-length or truncated LCoR as indicated ( C ). The luciferase activity was measured and is shown as relative luciferase units ( RLU ). D–F , 3T3-L1 cells were infected with retrovirus expressing a PPAR γ 2 promoter (−602 to +52)-driven luciferase reporter with or without retrovirus expressing F-LCoR ( D ), shLCoR ( E ), or truncated LCoR ( F ) as indicated. The luciferase activities were analyzed before or 48 h after differentiation induction using insulin, dexamethasone, and 3-isobutyl-1-methylxanthine ( IDM ) and shown as relative luciferase units ( RLU ). G and H , representative image ( G ) and percentages of BrdU-positive cells ( H ) are shown. 18 h after adipogenic induction, 3T3-L1 cells stably overexpressing F-LCoR or an empty vector (pMSCV) were labeled with BrdU for 2 h and then stained with DAPI. The fluorescence of BrdU ( green ) and DAPI ( blue ) was detected with a fluorescence microscope. I–L , 3T3-L1 cells stably overexpressing F-LCoR or pMSCV were harvested at the indicated times after adipogenic induction. The mRNA levels of CCND2 ( I ), CCNE1 ( J ), Gins1 ( K ), and Mcm3 ( L ) were measured by qPCR analysis. M , the efficiency of the knockdown of C/EBPβ in 3T3-L1 cells was determined by Western blotting. 3T3-L1 preadipocytes were infected with a retroviral vector containing shC/EBPβ or shLacZ as a control. N–Q , 3T3-L1 cells infected with shC/EBPβ or shLacZ were harvested at the indicated times after adipogenic induction. The mRNA levels of CCND2 ( N ), CCNE1 ( O ), Gins1 ( P ), and Mcm3 ( Q ) were measured by qPCR analysis. Blots shown here are representative of at least three independent experiments. Data are represented as mean ± S.D. ( n = 3–4). Error bars represent S.D. *, p

    Article Snippet: For adipogenic differentiation, 2-day postconfluent 3T3-L1 preadipocytes were induced by adipogenic differentiation medium containing high-glucose DMEM, 10% FBS, 1 μg/ml insulin (Sigma I1882), 1 μ m dexamethasone (Sigma D1756), and 0.5 m m 3-isobutyl-1-methylxanthine (Sigma I5879) for 2 days.

    Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Construct, Infection, Expressing, Stable Transfection, Labeling, Staining, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Western Blot

    PTH-induced urinary cAMP, phosphate excretion, and cAMP formation in renal cortical tubule suspensions are impaired in AC6 −/− mice. (A) Bolus application of PTH in WT mice dose-dependently increased fractional urinary P i excretion and urinary cAMP excretion. In contrast to WT mice, the phosphaturic effect and cAMP formation in response to PTH are absent in AC6 −/− mice. (B) Stimulation of cAMP formation by forskolin (Forsk; 10 µ mol/L) or PTH is absent in freshly isolated renal cortical tubule suspensions of AC6 −/− compared with WT mice. Protein concentration was adjusted to 60 µ g/vial. Experiments were performed with phosphodiesterase inhibition (0.5 mmol/L 3-isobutyl-1-methylxanthine; n =6–8/genotype for clearance experiments and n =5/genotype for isolated cortical tubules). bw, body weight. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Renal Phosphate Wasting in the Absence of Adenylyl Cyclase 6

    doi: 10.1681/ASN.2013101102

    Figure Lengend Snippet: PTH-induced urinary cAMP, phosphate excretion, and cAMP formation in renal cortical tubule suspensions are impaired in AC6 −/− mice. (A) Bolus application of PTH in WT mice dose-dependently increased fractional urinary P i excretion and urinary cAMP excretion. In contrast to WT mice, the phosphaturic effect and cAMP formation in response to PTH are absent in AC6 −/− mice. (B) Stimulation of cAMP formation by forskolin (Forsk; 10 µ mol/L) or PTH is absent in freshly isolated renal cortical tubule suspensions of AC6 −/− compared with WT mice. Protein concentration was adjusted to 60 µ g/vial. Experiments were performed with phosphodiesterase inhibition (0.5 mmol/L 3-isobutyl-1-methylxanthine; n =6–8/genotype for clearance experiments and n =5/genotype for isolated cortical tubules). bw, body weight. * P

    Article Snippet: Renal cortex was isolated using a modification of the method described by Guder, and cAMP accumulation was measured as described., Tubule suspensions were stimulated with PTH (1 nmol/L to 10 µ mol/L; GenScript), forskolin (10 µ mol/L; Ascent Scientific, Princeton, NJ), or vehicle for 15 minutes at 37°C in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (pretreated for 10 minutes, 0.5 mmol/L; Sigma-Aldrich, St. Louis, MO).

    Techniques: Mouse Assay, Isolation, Protein Concentration, Inhibition

    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