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  • 95
    Thermo Fisher 3 isobutyl 1 methylxanthine
    3 Isobutyl 1 Methylxanthine, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ibmx
    Ibmx, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3582 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ibmx  (Tocris)
    95
    Tocris ibmx
    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 <t>Fsk.</t> 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 <t>IBMX</t> 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
    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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    94
    Cayman Chemical 3 isobutyl 1 methylxanthine
    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 <t>Fsk.</t> 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 <t>IBMX</t> 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
    3 Isobutyl 1 Methylxanthine, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 94/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM 3 isobutyl 1 methylxanthine
    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 <t>Fsk.</t> 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 <t>IBMX</t> 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
    3 Isobutyl 1 Methylxanthine, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore isobutyl methyl xanthine ibmx
    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 <t>Fsk.</t> 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 <t>IBMX</t> 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
    Isobutyl Methyl Xanthine Ibmx, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore isobutyl 1 methylxanthine ibmx
    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 <t>Fsk.</t> 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 <t>IBMX</t> 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
    Isobutyl 1 Methylxanthine Ibmx, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore m 3 isobutyl 1 methylxanthine ibmx
    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 <t>Fsk.</t> 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 <t>IBMX</t> 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
    M 3 Isobutyl 1 Methylxanthine Ibmx, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH ibmx
    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 <t>Fsk.</t> 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 <t>IBMX</t> 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
    Ibmx, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Merck KGaA ibmx
    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 <t>Fsk.</t> 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 <t>IBMX</t> 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
    Ibmx, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Nacalai 3 isobutyl 1 methylxanthine
    Comparison of ASC differentiation to adipocytes. Comparison of adipocyte differentiation between the WT ( Pin1 +/+ ; p53 -/- ) ASC and Pin1-KO ( Pin1 -/- ; p53 -/- ) ASC, and Pin1-KO ASC rescued with the lentiviral Pin1 cDNA. The wild type ASC, the Pin1-KO ASC- infected with Mock and the Pin1-KO ASC- infected with lentiviral Pin1 cDNA were cultured in DMEM containing 0.5 mM <t>3-isobutyl-1-methylxanthine,</t> 1 μM dexamethasone, and 1.7 μM insulin for 0–16 days. ASCs were treated with 4% paraformaldehyde and 60% 2-propanol, and then stained with Oil Red O. The images of the oil red O-stained cells on 0, 4, 7, 10, 13, and 16 days after treatment with the differentiation reagent are shown.
    3 Isobutyl 1 Methylxanthine, supplied by Nacalai, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Applichem ibmx
    Activity measurements in HEK-TM cells. ( A ) <t>bPAC</t> activity measurements in HEK-TM cells. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca 2+ (HEK-TM). bPAC-mCherry was co-expressed with the mNphp3(201)-tagged mCherry nanobody. Light-dependent activation of bPAC increases intracellular cAMP levels, leading to a Ca 2+ influx, which was quantified using a fluorescent Ca 2+ dye (GFP-certified FluoForte). bPAC activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression with the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). bPAC activity was determined according to the maximum amplitude of the Ca 2+ signal after light stimulation (465 nm light pulse, 1 s, 162 µW/cm²) compared to the ionomycin-evoked Ca 2+ signal. 5 min before light stimulation, cells were treated with 25 µM of <t>IBMX</t> to inhibit phosphodiesterases and sustain a long-lasting increase in cAMP. NT: non-transfected cells. ( B ) LAPD activity measurements in HEK-TM cells. To measure LAPD activity, HEK-TM cells were pre-stimulated with 100 μM NKH477 to activate transmembrane adenylate cyclases (AC), thus increasing cAMP levels. Ca 2+ influx was detected by a Ca 2+ dye (Fluo4-AM). LAPD activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression of the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). Fluo4-AM-loaded HEK-TM cells were incubated with 100 μM NKH477 during continuous 850 nm light illumination (0.5 µW/cm²). When reaching a steady-state, light was switched to 690 nm (0.5 µW/cm²) to stimulate LAPD activity. LAPD activity was determined as the maximal decrease compared to the maximal Ca 2+ signal amplitude after NKH477 addition. Data are shown as individual data points (each data point represents and independent experiment and corresponds to the average of a duplicate or triplicate measurement) and mean ± S.D., p-values calculated using unpaired, two-sided Student's t-test compared to Sstr3-eGFP are indicated. All HEK-293 cells were non-ciliated.
    Ibmx, supplied by Applichem, used in various techniques. Bioz Stars score: 92/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ibmx free m16 medium
    Activity measurements in HEK-TM cells. ( A ) <t>bPAC</t> activity measurements in HEK-TM cells. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca 2+ (HEK-TM). bPAC-mCherry was co-expressed with the mNphp3(201)-tagged mCherry nanobody. Light-dependent activation of bPAC increases intracellular cAMP levels, leading to a Ca 2+ influx, which was quantified using a fluorescent Ca 2+ dye (GFP-certified FluoForte). bPAC activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression with the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). bPAC activity was determined according to the maximum amplitude of the Ca 2+ signal after light stimulation (465 nm light pulse, 1 s, 162 µW/cm²) compared to the ionomycin-evoked Ca 2+ signal. 5 min before light stimulation, cells were treated with 25 µM of <t>IBMX</t> to inhibit phosphodiesterases and sustain a long-lasting increase in cAMP. NT: non-transfected cells. ( B ) LAPD activity measurements in HEK-TM cells. To measure LAPD activity, HEK-TM cells were pre-stimulated with 100 μM NKH477 to activate transmembrane adenylate cyclases (AC), thus increasing cAMP levels. Ca 2+ influx was detected by a Ca 2+ dye (Fluo4-AM). LAPD activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression of the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). Fluo4-AM-loaded HEK-TM cells were incubated with 100 μM NKH477 during continuous 850 nm light illumination (0.5 µW/cm²). When reaching a steady-state, light was switched to 690 nm (0.5 µW/cm²) to stimulate LAPD activity. LAPD activity was determined as the maximal decrease compared to the maximal Ca 2+ signal amplitude after NKH477 addition. Data are shown as individual data points (each data point represents and independent experiment and corresponds to the average of a duplicate or triplicate measurement) and mean ± S.D., p-values calculated using unpaired, two-sided Student's t-test compared to Sstr3-eGFP are indicated. All HEK-293 cells were non-ciliated.
    Ibmx Free M16 Medium, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co 3 isobutyl 1 methylxanthine
    Activity measurements in HEK-TM cells. ( A ) <t>bPAC</t> activity measurements in HEK-TM cells. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca 2+ (HEK-TM). bPAC-mCherry was co-expressed with the mNphp3(201)-tagged mCherry nanobody. Light-dependent activation of bPAC increases intracellular cAMP levels, leading to a Ca 2+ influx, which was quantified using a fluorescent Ca 2+ dye (GFP-certified FluoForte). bPAC activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression with the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). bPAC activity was determined according to the maximum amplitude of the Ca 2+ signal after light stimulation (465 nm light pulse, 1 s, 162 µW/cm²) compared to the ionomycin-evoked Ca 2+ signal. 5 min before light stimulation, cells were treated with 25 µM of <t>IBMX</t> to inhibit phosphodiesterases and sustain a long-lasting increase in cAMP. NT: non-transfected cells. ( B ) LAPD activity measurements in HEK-TM cells. To measure LAPD activity, HEK-TM cells were pre-stimulated with 100 μM NKH477 to activate transmembrane adenylate cyclases (AC), thus increasing cAMP levels. Ca 2+ influx was detected by a Ca 2+ dye (Fluo4-AM). LAPD activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression of the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). Fluo4-AM-loaded HEK-TM cells were incubated with 100 μM NKH477 during continuous 850 nm light illumination (0.5 µW/cm²). When reaching a steady-state, light was switched to 690 nm (0.5 µW/cm²) to stimulate LAPD activity. LAPD activity was determined as the maximal decrease compared to the maximal Ca 2+ signal amplitude after NKH477 addition. Data are shown as individual data points (each data point represents and independent experiment and corresponds to the average of a duplicate or triplicate measurement) and mean ± S.D., p-values calculated using unpaired, two-sided Student's t-test compared to Sstr3-eGFP are indicated. All HEK-293 cells were non-ciliated.
    3 Isobutyl 1 Methylxanthine, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    IBMX is a general inhibitor of cyclic nucleotide phosphodiesterases increasing the level of intracellular cAMP It is used to increase cell differentiation decrease proliferation and induce apoptosis IBMX is reported
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    IBMX  (Cayman)
    N/A
    IBMX is a widely used non specific inhibitor of cyclic AMP and cyclic GMP phosphodiesterases PDEs IC 19 50 18 13 32 7 and 50 μM for PDE1 PDE2 PDE3
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    N/A
    Cell permeable A nonspecific phosphodiesterase inhibitor IC₅₀ values are 13 18 19 32 and 50 μM for PDE4 PDE3 PDE1 PDE5 and PDE2 respectively
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    Image Search Results


    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

    Comparison of ASC differentiation to adipocytes. Comparison of adipocyte differentiation between the WT ( Pin1 +/+ ; p53 -/- ) ASC and Pin1-KO ( Pin1 -/- ; p53 -/- ) ASC, and Pin1-KO ASC rescued with the lentiviral Pin1 cDNA. The wild type ASC, the Pin1-KO ASC- infected with Mock and the Pin1-KO ASC- infected with lentiviral Pin1 cDNA were cultured in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin for 0–16 days. ASCs were treated with 4% paraformaldehyde and 60% 2-propanol, and then stained with Oil Red O. The images of the oil red O-stained cells on 0, 4, 7, 10, 13, and 16 days after treatment with the differentiation reagent are shown.

    Journal: PLoS ONE

    Article Title: Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes

    doi: 10.1371/journal.pone.0168830

    Figure Lengend Snippet: Comparison of ASC differentiation to adipocytes. Comparison of adipocyte differentiation between the WT ( Pin1 +/+ ; p53 -/- ) ASC and Pin1-KO ( Pin1 -/- ; p53 -/- ) ASC, and Pin1-KO ASC rescued with the lentiviral Pin1 cDNA. The wild type ASC, the Pin1-KO ASC- infected with Mock and the Pin1-KO ASC- infected with lentiviral Pin1 cDNA were cultured in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin for 0–16 days. ASCs were treated with 4% paraformaldehyde and 60% 2-propanol, and then stained with Oil Red O. The images of the oil red O-stained cells on 0, 4, 7, 10, 13, and 16 days after treatment with the differentiation reagent are shown.

    Article Snippet: The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 0.5 mM 3-isobutyl-1-methylxanthine (Nacalai tesque), 1 μM dexamethasone (Sigma), and 1.7 μM insulin (Wako) with brown algae polyphenol (0–150 μg/ml) for a span of 16 days.

    Techniques: Infection, Cell Culture, Staining

    Effect of brown algae polyphenol on the differentiation of NIH3T3-L1 cells to adipocytes. A) The amount of the intracellular lipid stained with oil red O. NIH3T3-L1 cells were cultured in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin with 0 (diamond), 50 (square), 100 (triangle), and 150 μg/ml (x) brown algae polyphenol for 0–8 days. NIH3T3-L1 cells were treated with 4% paraformaldehyde and 60% 2-propanol and then stained with Oil Red O. The amount of oil red O extracted from the cells was determined three times by measuring absorbance at 550 nm (means ± SEM). B) PCR analysis of adipocyte biomarker mRNA levels. NIH3T3-L1 cells were cultured with 0, 50, 100, and 150 μg/ml of brown algae polyphenol for 0–8 days. The mRNA levels of PPARγ, C/EBPα, Glut4, FABP4, and LPL in these cells were compared by PCR.

    Journal: PLoS ONE

    Article Title: Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes

    doi: 10.1371/journal.pone.0168830

    Figure Lengend Snippet: Effect of brown algae polyphenol on the differentiation of NIH3T3-L1 cells to adipocytes. A) The amount of the intracellular lipid stained with oil red O. NIH3T3-L1 cells were cultured in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin with 0 (diamond), 50 (square), 100 (triangle), and 150 μg/ml (x) brown algae polyphenol for 0–8 days. NIH3T3-L1 cells were treated with 4% paraformaldehyde and 60% 2-propanol and then stained with Oil Red O. The amount of oil red O extracted from the cells was determined three times by measuring absorbance at 550 nm (means ± SEM). B) PCR analysis of adipocyte biomarker mRNA levels. NIH3T3-L1 cells were cultured with 0, 50, 100, and 150 μg/ml of brown algae polyphenol for 0–8 days. The mRNA levels of PPARγ, C/EBPα, Glut4, FABP4, and LPL in these cells were compared by PCR.

    Article Snippet: The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 0.5 mM 3-isobutyl-1-methylxanthine (Nacalai tesque), 1 μM dexamethasone (Sigma), and 1.7 μM insulin (Wako) with brown algae polyphenol (0–150 μg/ml) for a span of 16 days.

    Techniques: Staining, Cell Culture, Polymerase Chain Reaction, Biomarker Assay

    Activity measurements in HEK-TM cells. ( A ) bPAC activity measurements in HEK-TM cells. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca 2+ (HEK-TM). bPAC-mCherry was co-expressed with the mNphp3(201)-tagged mCherry nanobody. Light-dependent activation of bPAC increases intracellular cAMP levels, leading to a Ca 2+ influx, which was quantified using a fluorescent Ca 2+ dye (GFP-certified FluoForte). bPAC activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression with the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). bPAC activity was determined according to the maximum amplitude of the Ca 2+ signal after light stimulation (465 nm light pulse, 1 s, 162 µW/cm²) compared to the ionomycin-evoked Ca 2+ signal. 5 min before light stimulation, cells were treated with 25 µM of IBMX to inhibit phosphodiesterases and sustain a long-lasting increase in cAMP. NT: non-transfected cells. ( B ) LAPD activity measurements in HEK-TM cells. To measure LAPD activity, HEK-TM cells were pre-stimulated with 100 μM NKH477 to activate transmembrane adenylate cyclases (AC), thus increasing cAMP levels. Ca 2+ influx was detected by a Ca 2+ dye (Fluo4-AM). LAPD activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression of the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). Fluo4-AM-loaded HEK-TM cells were incubated with 100 μM NKH477 during continuous 850 nm light illumination (0.5 µW/cm²). When reaching a steady-state, light was switched to 690 nm (0.5 µW/cm²) to stimulate LAPD activity. LAPD activity was determined as the maximal decrease compared to the maximal Ca 2+ signal amplitude after NKH477 addition. Data are shown as individual data points (each data point represents and independent experiment and corresponds to the average of a duplicate or triplicate measurement) and mean ± S.D., p-values calculated using unpaired, two-sided Student's t-test compared to Sstr3-eGFP are indicated. All HEK-293 cells were non-ciliated.

    Journal: eLife

    Article Title: Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

    doi: 10.7554/eLife.57907

    Figure Lengend Snippet: Activity measurements in HEK-TM cells. ( A ) bPAC activity measurements in HEK-TM cells. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca 2+ (HEK-TM). bPAC-mCherry was co-expressed with the mNphp3(201)-tagged mCherry nanobody. Light-dependent activation of bPAC increases intracellular cAMP levels, leading to a Ca 2+ influx, which was quantified using a fluorescent Ca 2+ dye (GFP-certified FluoForte). bPAC activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression with the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). bPAC activity was determined according to the maximum amplitude of the Ca 2+ signal after light stimulation (465 nm light pulse, 1 s, 162 µW/cm²) compared to the ionomycin-evoked Ca 2+ signal. 5 min before light stimulation, cells were treated with 25 µM of IBMX to inhibit phosphodiesterases and sustain a long-lasting increase in cAMP. NT: non-transfected cells. ( B ) LAPD activity measurements in HEK-TM cells. To measure LAPD activity, HEK-TM cells were pre-stimulated with 100 μM NKH477 to activate transmembrane adenylate cyclases (AC), thus increasing cAMP levels. Ca 2+ influx was detected by a Ca 2+ dye (Fluo4-AM). LAPD activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression of the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). Fluo4-AM-loaded HEK-TM cells were incubated with 100 μM NKH477 during continuous 850 nm light illumination (0.5 µW/cm²). When reaching a steady-state, light was switched to 690 nm (0.5 µW/cm²) to stimulate LAPD activity. LAPD activity was determined as the maximal decrease compared to the maximal Ca 2+ signal amplitude after NKH477 addition. Data are shown as individual data points (each data point represents and independent experiment and corresponds to the average of a duplicate or triplicate measurement) and mean ± S.D., p-values calculated using unpaired, two-sided Student's t-test compared to Sstr3-eGFP are indicated. All HEK-293 cells were non-ciliated.

    Article Snippet: During bPAC activity measurements, cells were stimulated with a 488-nm-light pulse (1 W/cm²) for 1 s at 3 min, for 10 s at 21 min, and for 60 s at 45 min. For bPAC+nanobody activity measurements, cells were incubated with 25 µM of IBMX (stock: 250 mM in DMSO, AppliChem) from 5 min before light activation on; light activation was induced 2 min after starting the recording using a 488-nm-light pulse (1 s, 162 µW/cm²).

    Techniques: Activity Assay, Binding Assay, Activation Assay, Laser Capture Microdissection, Expressing, Negative Control, Transfection, Incubation

    Functional characterization of bPAC in the cell body and cilium. ( A ) Schematic overview of the bPAC activity assay in non-ciliated HEK293 cells using R-FlincA (see B-C ). ( B ) HEK293 cells were transfected with bPAC-eGFP and R-FlincA or the non-binding R-FlincA mutant ( Ohta et al., 2018 ). The change in R-FlincA fluorescence was measured over time before and after photoactivation of bPAC (5 s, white light, 2.1 mW/cm 2 at 480 nm). Data are shown as mean (solid lines)± S.D. (dotted lines), n = 3 with 4 cells per experiment. ( C ) Normalized R-FlincA or R-FlincA mutant fluorescence directly before and for the maximal amplitude after photoactivation. Data extracted from C; p-values have been calculated using a paired, two-sided Student’s t-test. ( D ) Schematic overview of the assay to measure ciliary cAMP dynamics using 5-HT 6 -mCherry-cADDis after pharmacologically increasing cAMP levels (see E-F). ( E ) Ciliary cAMP dynamics measured using 5-HT 6 -mCherry-cADDis. Cells were stimulated with 250 μM IBMX (light blue) or 40 μM Forskolin (purple). The normalized ratio of ciliary mCherry/cpEGFP fluorescence is shown as mean (solid lines)± S.D. (dotted lines); p-values have been calculated by paired, two-sided Student’s t-test. ( F ) Mean change in the normalized ratio of ciliary mCherry/cpEGFP fluorescence 60–120 s after stimulation with buffer, IBMX, or Forskolin. Data are shown as individual data points, the mean ± S.D. is indicated; p-values have been calculated by a two-sided Mann-Whitney test. ( G ) Schematic overview of the assay to measure light-evoked ciliary cAMP dynamics after bPAC stimulation using 5-HT 6 -cADDis (see H-I). ( H ) 5-HT6-cADDis fluorescence in cilia with mNphp3(201)-VHH LaM-2 -HA targeted mCherry or bPAC-mCherry in the first frame of imaging. Scale bar: 2 μm. ( I ) Mean normalized ciliary cpEGFP fluorescence in the first frame. All data have been normalized to the mean cpEGFP fluorescence in the mCherry control. Data are shown as individual data points, the mean ± S.D. is indicated; p-values have been calculated by unpaired, two-sided Student’s t-test.

    Journal: eLife

    Article Title: Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

    doi: 10.7554/eLife.57907

    Figure Lengend Snippet: Functional characterization of bPAC in the cell body and cilium. ( A ) Schematic overview of the bPAC activity assay in non-ciliated HEK293 cells using R-FlincA (see B-C ). ( B ) HEK293 cells were transfected with bPAC-eGFP and R-FlincA or the non-binding R-FlincA mutant ( Ohta et al., 2018 ). The change in R-FlincA fluorescence was measured over time before and after photoactivation of bPAC (5 s, white light, 2.1 mW/cm 2 at 480 nm). Data are shown as mean (solid lines)± S.D. (dotted lines), n = 3 with 4 cells per experiment. ( C ) Normalized R-FlincA or R-FlincA mutant fluorescence directly before and for the maximal amplitude after photoactivation. Data extracted from C; p-values have been calculated using a paired, two-sided Student’s t-test. ( D ) Schematic overview of the assay to measure ciliary cAMP dynamics using 5-HT 6 -mCherry-cADDis after pharmacologically increasing cAMP levels (see E-F). ( E ) Ciliary cAMP dynamics measured using 5-HT 6 -mCherry-cADDis. Cells were stimulated with 250 μM IBMX (light blue) or 40 μM Forskolin (purple). The normalized ratio of ciliary mCherry/cpEGFP fluorescence is shown as mean (solid lines)± S.D. (dotted lines); p-values have been calculated by paired, two-sided Student’s t-test. ( F ) Mean change in the normalized ratio of ciliary mCherry/cpEGFP fluorescence 60–120 s after stimulation with buffer, IBMX, or Forskolin. Data are shown as individual data points, the mean ± S.D. is indicated; p-values have been calculated by a two-sided Mann-Whitney test. ( G ) Schematic overview of the assay to measure light-evoked ciliary cAMP dynamics after bPAC stimulation using 5-HT 6 -cADDis (see H-I). ( H ) 5-HT6-cADDis fluorescence in cilia with mNphp3(201)-VHH LaM-2 -HA targeted mCherry or bPAC-mCherry in the first frame of imaging. Scale bar: 2 μm. ( I ) Mean normalized ciliary cpEGFP fluorescence in the first frame. All data have been normalized to the mean cpEGFP fluorescence in the mCherry control. Data are shown as individual data points, the mean ± S.D. is indicated; p-values have been calculated by unpaired, two-sided Student’s t-test.

    Article Snippet: During bPAC activity measurements, cells were stimulated with a 488-nm-light pulse (1 W/cm²) for 1 s at 3 min, for 10 s at 21 min, and for 60 s at 45 min. For bPAC+nanobody activity measurements, cells were incubated with 25 µM of IBMX (stock: 250 mM in DMSO, AppliChem) from 5 min before light activation on; light activation was induced 2 min after starting the recording using a 488-nm-light pulse (1 s, 162 µW/cm²).

    Techniques: Functional Assay, Activity Assay, Transfection, Binding Assay, Mutagenesis, Fluorescence, MANN-WHITNEY, Laser Capture Microdissection, Imaging

    Characterization of the ciliary-targeted cAMP mlCNBD-FRET biosensor. ( A ) Localization of mNphp3(201)-mlCNBD-FRET to primary cilia in mIMCD-3 cells. ( B ) FRET imaging in non-ciliated HEK293 cells expressing mlCNBD-FRET or mNphp3(201)-mlCNBD-FRET under control conditions or after stimulation with 20 μM isoproterenol (addition depicted with dotted line). Data are shown as mean (n = 3 independent experiments, 30–90 cells per experiment). Schematic overview of mlCNBD-FRET imaging is shown on the left. ( C ) Comparison of the maximal FRET change for data shown in B. Data are presented as individual data points and mean ± S.D.; p-value calculated using a two-tailed Mann-Whitney test is indicated. ( D ) Localization of mNphp3(201)-VHH enhancer -mCherry in HEK293 cells. The box indicates the position of the magnified view shown at the bottom for the individual channels. ( E./F ) Localization of mlCNBD-FRET in HEK293 cells in the presence (E) or absence (F) of mNphp3(201)-VHH enhancer -mCherry. In F, mCherry only was used as a control. Scale bar: 10 μm. The box in indicates the position of the magnified view shown at the bottom. left: overlay; middle and right: individual channels. ( G ) HEK293 cells were transfected with cerulean or citrine in the presence of mCherry (control) or the eGFP nanobody mNphp3(201)-VHH enhancer -mCherry. Fluorescence intensities of cerulean or citrine were normalized to the mCherry fluorescence of the nanobody or mCherry only in the same cell, and the relative change in fluorescence compared to the control condition (mCherry only) was plotted. Data are shown as mean ± S.D., n = 3–6 with 2–30 cells per experiment; p-values were determined using an unpaired, two-sided Student’s t-test. ( H ) FRET imaging in primary cilia of mIMCD-3 cells expressing mlCNBD-FRET and mNphp3(201)-VHH enhancer -mCherry. Cells have been stimulated with 250 μm IBMX (top) or buffer only (bottom). Images show the change in cerulean/citrine ratio (color-scheme indicated on the right) from different time points (top) of Video 1 . Scale bar: 2 μm.

    Journal: eLife

    Article Title: Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

    doi: 10.7554/eLife.57907

    Figure Lengend Snippet: Characterization of the ciliary-targeted cAMP mlCNBD-FRET biosensor. ( A ) Localization of mNphp3(201)-mlCNBD-FRET to primary cilia in mIMCD-3 cells. ( B ) FRET imaging in non-ciliated HEK293 cells expressing mlCNBD-FRET or mNphp3(201)-mlCNBD-FRET under control conditions or after stimulation with 20 μM isoproterenol (addition depicted with dotted line). Data are shown as mean (n = 3 independent experiments, 30–90 cells per experiment). Schematic overview of mlCNBD-FRET imaging is shown on the left. ( C ) Comparison of the maximal FRET change for data shown in B. Data are presented as individual data points and mean ± S.D.; p-value calculated using a two-tailed Mann-Whitney test is indicated. ( D ) Localization of mNphp3(201)-VHH enhancer -mCherry in HEK293 cells. The box indicates the position of the magnified view shown at the bottom for the individual channels. ( E./F ) Localization of mlCNBD-FRET in HEK293 cells in the presence (E) or absence (F) of mNphp3(201)-VHH enhancer -mCherry. In F, mCherry only was used as a control. Scale bar: 10 μm. The box in indicates the position of the magnified view shown at the bottom. left: overlay; middle and right: individual channels. ( G ) HEK293 cells were transfected with cerulean or citrine in the presence of mCherry (control) or the eGFP nanobody mNphp3(201)-VHH enhancer -mCherry. Fluorescence intensities of cerulean or citrine were normalized to the mCherry fluorescence of the nanobody or mCherry only in the same cell, and the relative change in fluorescence compared to the control condition (mCherry only) was plotted. Data are shown as mean ± S.D., n = 3–6 with 2–30 cells per experiment; p-values were determined using an unpaired, two-sided Student’s t-test. ( H ) FRET imaging in primary cilia of mIMCD-3 cells expressing mlCNBD-FRET and mNphp3(201)-VHH enhancer -mCherry. Cells have been stimulated with 250 μm IBMX (top) or buffer only (bottom). Images show the change in cerulean/citrine ratio (color-scheme indicated on the right) from different time points (top) of Video 1 . Scale bar: 2 μm.

    Article Snippet: During bPAC activity measurements, cells were stimulated with a 488-nm-light pulse (1 W/cm²) for 1 s at 3 min, for 10 s at 21 min, and for 60 s at 45 min. For bPAC+nanobody activity measurements, cells were incubated with 25 µM of IBMX (stock: 250 mM in DMSO, AppliChem) from 5 min before light activation on; light activation was induced 2 min after starting the recording using a 488-nm-light pulse (1 s, 162 µW/cm²).

    Techniques: Imaging, Expressing, Two Tailed Test, MANN-WHITNEY, Transfection, Fluorescence