ibmx  (Millipore)


Bioz Verified Symbol Millipore is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    3 Isobutyl 1 methylxanthine
    Description:

    Catalog Number:
    i7018
    Price:
    None
    Applications:
    3-Isobutyl-1-methylxanthine has been used:. for adipogenic differentiation. in tissue culture. 3T3-L1 preadipocyte differentiation(
    Buy from Supplier


    Structured Review

    Millipore ibmx
    3 Isobutyl 1 methylxanthine

    https://www.bioz.com/result/ibmx/product/Millipore
    Average 99 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    ibmx - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Constitutively active phosphodiesterase activity regulates urinary bladder smooth muscle function: critical role of KCa1.1 channel"

    Article Title: Constitutively active phosphodiesterase activity regulates urinary bladder smooth muscle function: critical role of KCa1.1 channel

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00351.2012

    Pharmacological inhibition of K Ca 1.1 channels with paxilline abolished the hyperpolarizing effect of IBMX in human UBSM-isolated cells. A : original current-clamp recording illustrating that 1 μM paxilline abolished STHs and that the subsequent
    Figure Legend Snippet: Pharmacological inhibition of K Ca 1.1 channels with paxilline abolished the hyperpolarizing effect of IBMX in human UBSM-isolated cells. A : original current-clamp recording illustrating that 1 μM paxilline abolished STHs and that the subsequent

    Techniques Used: Inhibition, Isolation

    2) Product Images from "Isolation of alveolar epithelial type II progenitor cells from adult human lungs"

    Article Title: Isolation of alveolar epithelial type II progenitor cells from adult human lungs

    Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

    doi: 10.1038/labinvest.2010.187

    The pro-SP-C + /CD90 + cells differentiate into ATII cells in vitro . ( a – f ) Representative morphologic appearances of cells cultured on a mixture of Matrigel and rat tail collagen with keratinocyte growth factor (KGF), cAMP, and IBMX (KIA(+), left side panels) and without any additives (KIA(−), right side panels) in phase contrast images ( a , b ) and hematoxylin–eosin (HE) staining ( c , d ) and immunofluorescence staining of pro-SP-C (green) and CD90 (red) ( e , f ). White arrowheads indicate pro-SP-C + /CD90 − cells (ATII cells) and white arrows show SP-C − /CD90 + cells ( e , f ). A pro-SP-C + /CD90 + cell was still observed (yellow arrowhead in e ). Unidentified differentiated cells expressing neither pro-SP-C nor CD90 were seen (yellow arrows in e , f ). ( g , h ) Transmission electron microscopic images of differentiated cells in KIA(+) group. ( g ) Lamellar bodies (a white-framed rectangle) and apical microvilli (arrows) were observed as distinctive structures in ATII cells. ( h ) A higher magnification of lamellar bodies from a white-framed rectangle in ( g ). ( i ) The ratio of the number pro-SP-C + /CD90 − cells to the number of DAPI-stained nuclei was significantly higher in the KIA(+) group than that in the KIA(−) group (47.8±12.2% vs 2.5±2.5%, n =4 in each, P =0.0003). ( j ) Representative RT–PCR analysis of SP-A, SP-C (ATII cell markers) and aquaporin 5 (AQP5; an ATI cell marker) and vimentin and α -SMA (mesenchymal markers) before and after differentiation. Scale bars: ( a , b ) 200 μ m; ( c , d ) 50 μ m; ( e , f ) 20 μ m; ( g ) 2 μ m; ( h ) 300 nm.
    Figure Legend Snippet: The pro-SP-C + /CD90 + cells differentiate into ATII cells in vitro . ( a – f ) Representative morphologic appearances of cells cultured on a mixture of Matrigel and rat tail collagen with keratinocyte growth factor (KGF), cAMP, and IBMX (KIA(+), left side panels) and without any additives (KIA(−), right side panels) in phase contrast images ( a , b ) and hematoxylin–eosin (HE) staining ( c , d ) and immunofluorescence staining of pro-SP-C (green) and CD90 (red) ( e , f ). White arrowheads indicate pro-SP-C + /CD90 − cells (ATII cells) and white arrows show SP-C − /CD90 + cells ( e , f ). A pro-SP-C + /CD90 + cell was still observed (yellow arrowhead in e ). Unidentified differentiated cells expressing neither pro-SP-C nor CD90 were seen (yellow arrows in e , f ). ( g , h ) Transmission electron microscopic images of differentiated cells in KIA(+) group. ( g ) Lamellar bodies (a white-framed rectangle) and apical microvilli (arrows) were observed as distinctive structures in ATII cells. ( h ) A higher magnification of lamellar bodies from a white-framed rectangle in ( g ). ( i ) The ratio of the number pro-SP-C + /CD90 − cells to the number of DAPI-stained nuclei was significantly higher in the KIA(+) group than that in the KIA(−) group (47.8±12.2% vs 2.5±2.5%, n =4 in each, P =0.0003). ( j ) Representative RT–PCR analysis of SP-A, SP-C (ATII cell markers) and aquaporin 5 (AQP5; an ATI cell marker) and vimentin and α -SMA (mesenchymal markers) before and after differentiation. Scale bars: ( a , b ) 200 μ m; ( c , d ) 50 μ m; ( e , f ) 20 μ m; ( g ) 2 μ m; ( h ) 300 nm.

    Techniques Used: In Vitro, Cell Culture, Staining, Immunofluorescence, Expressing, Transmission Assay, Reverse Transcription Polymerase Chain Reaction, Marker

    3) Product Images from "Intracellular potentiation between two second messenger systems may contribute to cholera toxin induced intestinal secretion in humans"

    Article Title: Intracellular potentiation between two second messenger systems may contribute to cholera toxin induced intestinal secretion in humans

    Journal: Gut

    doi:

    Stimulation of cAMP production in T 84 cells. cAMP levels (pmol/cm 2 of monolayer) were measured at 180 minutes after exposure to cholera toxin (CT) or 3-isobutyl-1-methylxanthine (IBMX, controls), and at 30 minutes after exposure to acetylcholine (ACh). When given in combination, ACh was added to CT after 150 minutes and cAMP levels were measured at 180 minutes. Barium (5 mM) was given 20 minutes before CT (0.01 μg/ml). Data are means (SEM) (n = 6).
    Figure Legend Snippet: Stimulation of cAMP production in T 84 cells. cAMP levels (pmol/cm 2 of monolayer) were measured at 180 minutes after exposure to cholera toxin (CT) or 3-isobutyl-1-methylxanthine (IBMX, controls), and at 30 minutes after exposure to acetylcholine (ACh). When given in combination, ACh was added to CT after 150 minutes and cAMP levels were measured at 180 minutes. Barium (5 mM) was given 20 minutes before CT (0.01 μg/ml). Data are means (SEM) (n = 6).

    Techniques Used:

    4) Product Images from "Inhibition of volume-regulated anion channels by expression of the cystic fibrosis transmembrane conductance regulator"

    Article Title: Inhibition of volume-regulated anion channels by expression of the cystic fibrosis transmembrane conductance regulator

    Journal: The Journal of Physiology

    doi: 10.1111/j.1469-7793.1999.075ad.x

    Volume-activated current in a COS cells overexpressing WT CFTR A , time course of the activation of I Cl,swell at +50 mV (○) and -80 mV (▵) in a non-transfected COS cell, during a superfusion with 25 % hypotonic solution (HTS). I-V relations were obtained at the time points indicated with filled symbols. B , time course of activation of I Cl,swell in a COS cell expressing CFTR, during superfusion with 25 % HTS. As a positive control for successful transfection, CFTR was activated with a cocktail of IBMX and forskolin dissolved in isotonic solution (I/F, horizontal bar) before or after application of HTS. I-V relations were obtained at time points indicated with filled symbols.
    Figure Legend Snippet: Volume-activated current in a COS cells overexpressing WT CFTR A , time course of the activation of I Cl,swell at +50 mV (○) and -80 mV (▵) in a non-transfected COS cell, during a superfusion with 25 % hypotonic solution (HTS). I-V relations were obtained at the time points indicated with filled symbols. B , time course of activation of I Cl,swell in a COS cell expressing CFTR, during superfusion with 25 % HTS. As a positive control for successful transfection, CFTR was activated with a cocktail of IBMX and forskolin dissolved in isotonic solution (I/F, horizontal bar) before or after application of HTS. I-V relations were obtained at time points indicated with filled symbols.

    Techniques Used: Activation Assay, Transfection, Expressing, Positive Control

    Volume-activated current in CPAE cells A , time course of activation of I Cl,swell in a non-transfected CPAE cell, measured at +50 mV (○) and -80 mV (▵), during superfusion with 25 % hypotonic solution (HTS; horizontal bar). I-V relations were obtained at indicated time points. B , time course of activation of I Cl,swell in a CPAE cell expressing WT CFTR. As a positive control for successful transfection, CFTR was activated with a cocktail of IBMX and forskolin (I/F, horizontal bar) dissolved in isotonic solution before or after application of HTS.
    Figure Legend Snippet: Volume-activated current in CPAE cells A , time course of activation of I Cl,swell in a non-transfected CPAE cell, measured at +50 mV (○) and -80 mV (▵), during superfusion with 25 % hypotonic solution (HTS; horizontal bar). I-V relations were obtained at indicated time points. B , time course of activation of I Cl,swell in a CPAE cell expressing WT CFTR. As a positive control for successful transfection, CFTR was activated with a cocktail of IBMX and forskolin (I/F, horizontal bar) dissolved in isotonic solution before or after application of HTS.

    Techniques Used: Activation Assay, Transfection, Expressing, Positive Control

    Volume-activated current co-activated with WT CFTR A , time course of activation of WT CFTR expressed in a CPAE cell, at +50 mV (○) and -80 mV (▵). Forskolin (0.7 μM) and (IBMX) 70 μM were applied (I/F, horizontal bar). B , time course of co-activation of WT CFTR and I Cl,swell as in A. When the CFTR current reached it's maximum, 25 % hypotonic solution (HTS, horizontal bar) was applied. I-V relations were obtained at the time points marked with filled symbols, from a linear ramp protocol from -100 to +50 mV. Holding potential was 0 mV.
    Figure Legend Snippet: Volume-activated current co-activated with WT CFTR A , time course of activation of WT CFTR expressed in a CPAE cell, at +50 mV (○) and -80 mV (▵). Forskolin (0.7 μM) and (IBMX) 70 μM were applied (I/F, horizontal bar). B , time course of co-activation of WT CFTR and I Cl,swell as in A. When the CFTR current reached it's maximum, 25 % hypotonic solution (HTS, horizontal bar) was applied. I-V relations were obtained at the time points marked with filled symbols, from a linear ramp protocol from -100 to +50 mV. Holding potential was 0 mV.

    Techniques Used: Activation Assay

    Volume-activated current in a CPAE cell transfected with ΔF508 CFTR A , time course of activation of I Cl,swell at +50 (○) and -80 mV (▵) in a transfected cell that did not respond to IBMX and forskolin (I/F, horizontal bar). Twenty-five per cent hypotonic solution was applied (HTS, horizontal bar). I-V relations were obtained at the indicated time points. B , time course of activation of I Cl,swell in a transfected cell that responds to application of IBMX and forskolin (horizontal bar). Twenty-five per cent HTS was applied before or after activation of ΔF508 CFTR (horizontal bar).
    Figure Legend Snippet: Volume-activated current in a CPAE cell transfected with ΔF508 CFTR A , time course of activation of I Cl,swell at +50 (○) and -80 mV (▵) in a transfected cell that did not respond to IBMX and forskolin (I/F, horizontal bar). Twenty-five per cent hypotonic solution was applied (HTS, horizontal bar). I-V relations were obtained at the indicated time points. B , time course of activation of I Cl,swell in a transfected cell that responds to application of IBMX and forskolin (horizontal bar). Twenty-five per cent HTS was applied before or after activation of ΔF508 CFTR (horizontal bar).

    Techniques Used: Transfection, Activation Assay

    Measurement of cell height changes in WT CFTR transfected and non-transfected CPAE cells during a 25 % hypotonic stimulus A , cell height measurement from WT CFTR transfected ( n = 5) and non-transfected CPAE cells ( n = 5) during application of 25 % hypotonic solution (HTS). B , cell height measurement from WT CFTR transfected ( n = 4) and non-transfected CPAE cells ( n = 4) during application of 25 % HTS plus CFTR activation cocktail (70 μM IBMX and 0.7 μM forskolin, I/F). ▵, non-transfected cells; ○, WT CFTR transfected cells. Values were normalized to the mean cell height in isotonic solution.
    Figure Legend Snippet: Measurement of cell height changes in WT CFTR transfected and non-transfected CPAE cells during a 25 % hypotonic stimulus A , cell height measurement from WT CFTR transfected ( n = 5) and non-transfected CPAE cells ( n = 5) during application of 25 % hypotonic solution (HTS). B , cell height measurement from WT CFTR transfected ( n = 4) and non-transfected CPAE cells ( n = 4) during application of 25 % HTS plus CFTR activation cocktail (70 μM IBMX and 0.7 μM forskolin, I/F). ▵, non-transfected cells; ○, WT CFTR transfected cells. Values were normalized to the mean cell height in isotonic solution.

    Techniques Used: Transfection, Activation Assay

    Representative current traces and I-V curves of WT CFTR and I Cl,swell in transfected and control cells A , current traces and I-V curve for I Cl,swell in a non-transfected CPAE cell, after application of 25 % hypotonic solution (HTS). Step protocol consisted of 1 s voltage steps ranging from -100 to +120 mV from a holding potential of 0 mV. Arrows indicate zero current level. B , current traces and I-V curve for CFTR in a WT CFTR transfected CPAE cell, after application of 100 μM IBMX and 1 μM forskolin dissolved in isotonic solution. Step protocol analogous to A , except for a pre-step of 300 ms to 0 mV. C , current traces and I-V curve for I Cl,swell in a WT CFTR transfected CPAE cell, after application of 25 % HTS. Step protocol analogous to A .
    Figure Legend Snippet: Representative current traces and I-V curves of WT CFTR and I Cl,swell in transfected and control cells A , current traces and I-V curve for I Cl,swell in a non-transfected CPAE cell, after application of 25 % hypotonic solution (HTS). Step protocol consisted of 1 s voltage steps ranging from -100 to +120 mV from a holding potential of 0 mV. Arrows indicate zero current level. B , current traces and I-V curve for CFTR in a WT CFTR transfected CPAE cell, after application of 100 μM IBMX and 1 μM forskolin dissolved in isotonic solution. Step protocol analogous to A , except for a pre-step of 300 ms to 0 mV. C , current traces and I-V curve for I Cl,swell in a WT CFTR transfected CPAE cell, after application of 25 % HTS. Step protocol analogous to A .

    Techniques Used: Transfection, Mass Spectrometry

    5) Product Images from "Membrane estrogen receptor-? levels in MCF-7 breast cancer cells predict cAMP and proliferation responses"

    Article Title: Membrane estrogen receptor-? levels in MCF-7 breast cancer cells predict cAMP and proliferation responses

    Journal: Breast Cancer Research

    doi: 10.1186/bcr958

    Kinetics of cAMP decrease in MCF-7 cells enriched for membrane estrogen receptor-α (mER high ) cells. (a) Cells were treated with 1 pmol/l 17β-estradiol (E 2 ) for different time intervals at 37°C in the presence of 1 mmol/l 3-isobutyl-1-methylxanthine (IBMX). The intracellular cAMP (circles) and that in the medium (squares) from the same cells were assessed. (b) cAMP was produced by directly stimulating adenylyl cyclase with 10 μmol/l forskolin for 15 min at 37°C. The decrease in cAMP in the cytosol was tested at 37°C in the absence (open circles) and presence of 1 mmol/l IBMX (closed circles). The entire regression lines were compared by evaluating the differences between the sums of squares of the residuals of individual lines with the sum of squares of the residuals of the combined line using an F-test. The data are presented as means ± standard error. The regression lines were significantly different ( P = 0.0001).
    Figure Legend Snippet: Kinetics of cAMP decrease in MCF-7 cells enriched for membrane estrogen receptor-α (mER high ) cells. (a) Cells were treated with 1 pmol/l 17β-estradiol (E 2 ) for different time intervals at 37°C in the presence of 1 mmol/l 3-isobutyl-1-methylxanthine (IBMX). The intracellular cAMP (circles) and that in the medium (squares) from the same cells were assessed. (b) cAMP was produced by directly stimulating adenylyl cyclase with 10 μmol/l forskolin for 15 min at 37°C. The decrease in cAMP in the cytosol was tested at 37°C in the absence (open circles) and presence of 1 mmol/l IBMX (closed circles). The entire regression lines were compared by evaluating the differences between the sums of squares of the residuals of individual lines with the sum of squares of the residuals of the combined line using an F-test. The data are presented as means ± standard error. The regression lines were significantly different ( P = 0.0001).

    Techniques Used: Produced

    6) Product Images from "CREB Is One Component of the Binding Complex of the Ces-2/E2A-HLF Binding Element and Is an Integral Part of the Interleukin-3 Survival Signal"

    Article Title: CREB Is One Component of the Binding Complex of the Ces-2/E2A-HLF Binding Element and Is an Integral Part of the Interleukin-3 Survival Signal

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.14.4636-4646.2001

    Transcriptional activation of CBE reporter genes by the cAMP/PKA/CREB signaling pathway. (A) Activation of CBE reporter genes by cAMP-elevating agents. 293T and PC12 cells were transfected with p3xCBE-Luc (1 μg) and treated with or without forskolin plus IBMX (as indicated at the bottom). (B) CREB-dependent activation of the CBE reporter gene by PKA in PC12 cells. PC12 cells were transfected with the PKAc expression plasmid (0.1 μg) and various luciferase reporter plasmids (1 μg of each), together with dominant negative mutant CREBR287L (1 μg). (C) CREB-dependent activation of CBE reporter genes by PKA in Ba/F3 cells. Ba/F3 cells were electroporated with the PKAc expression plasmid (0.45 μg) and CBE luciferase reporter plasmid (3 μg), together with CREBR287L (0, 1.5 and 4 μg; shown as a triangle). Twenty hours after transfection, cell lysates were prepared and analyzed by luciferase assays. Data shown are representative results from three independent experiments performed in duplicate (in PC12 cells) or quadruplicate (in Ba/F3 cells). Luciferase activities are plotted in arbitrary units per microgram of total protein.
    Figure Legend Snippet: Transcriptional activation of CBE reporter genes by the cAMP/PKA/CREB signaling pathway. (A) Activation of CBE reporter genes by cAMP-elevating agents. 293T and PC12 cells were transfected with p3xCBE-Luc (1 μg) and treated with or without forskolin plus IBMX (as indicated at the bottom). (B) CREB-dependent activation of the CBE reporter gene by PKA in PC12 cells. PC12 cells were transfected with the PKAc expression plasmid (0.1 μg) and various luciferase reporter plasmids (1 μg of each), together with dominant negative mutant CREBR287L (1 μg). (C) CREB-dependent activation of CBE reporter genes by PKA in Ba/F3 cells. Ba/F3 cells were electroporated with the PKAc expression plasmid (0.45 μg) and CBE luciferase reporter plasmid (3 μg), together with CREBR287L (0, 1.5 and 4 μg; shown as a triangle). Twenty hours after transfection, cell lysates were prepared and analyzed by luciferase assays. Data shown are representative results from three independent experiments performed in duplicate (in PC12 cells) or quadruplicate (in Ba/F3 cells). Luciferase activities are plotted in arbitrary units per microgram of total protein.

    Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Dominant Negative Mutation

    IL-3 induces CREB phosphorylation and survival function partly via the PKA pathway. (A) IL-3 induces PKA-dependent CREB Ser 133 phosphorylation. Cytokine-starved Ba/F3 cells were pretreated with dimethyl sulfoxide (lanes 1 and 2) or PKI (lanes 3 and 4) for 30 min before being restimulated with IL-3 (IL3). After 5 min of stimulation, cells were harvested and analyzed by Western blot analysis with antibodies specific to phosphorylated CREB at Ser 133 (-CREB) and to all forms of CREB (CREB). The star indicates the position of the phosphorylated ATF-2. (B) Increase of survival of Ba/F3 cells by cAMP-elevating agents. Viable cell number was measured by trypan blue staining after 18 h of incubation in cytokine-free medium without (−) or with (+) forskolin (FsK 100 μM) plus IBMX. Numbers of viable cells are presented as percentages of the number of cells initially seeded. Results shown are means ± standard deviations from two independent experiments performed in duplicate. (C) Decrease of cell viability by PKI. Ba/F3 cells were cultured in IL-3-containing medium supplemented with various doses of Myr-PKI (lanes 2 and 3, 10 and 20 μM). Cells cultured in cytokine-free medium were included as a negative control (lane 4). Numbers of viable cells under various treatments were determined and are presented as a percentage of the number of cells initially seeded. Shown are representative results from three independent experiments performed in duplicate.
    Figure Legend Snippet: IL-3 induces CREB phosphorylation and survival function partly via the PKA pathway. (A) IL-3 induces PKA-dependent CREB Ser 133 phosphorylation. Cytokine-starved Ba/F3 cells were pretreated with dimethyl sulfoxide (lanes 1 and 2) or PKI (lanes 3 and 4) for 30 min before being restimulated with IL-3 (IL3). After 5 min of stimulation, cells were harvested and analyzed by Western blot analysis with antibodies specific to phosphorylated CREB at Ser 133 (-CREB) and to all forms of CREB (CREB). The star indicates the position of the phosphorylated ATF-2. (B) Increase of survival of Ba/F3 cells by cAMP-elevating agents. Viable cell number was measured by trypan blue staining after 18 h of incubation in cytokine-free medium without (−) or with (+) forskolin (FsK 100 μM) plus IBMX. Numbers of viable cells are presented as percentages of the number of cells initially seeded. Results shown are means ± standard deviations from two independent experiments performed in duplicate. (C) Decrease of cell viability by PKI. Ba/F3 cells were cultured in IL-3-containing medium supplemented with various doses of Myr-PKI (lanes 2 and 3, 10 and 20 μM). Cells cultured in cytokine-free medium were included as a negative control (lane 4). Numbers of viable cells under various treatments were determined and are presented as a percentage of the number of cells initially seeded. Shown are representative results from three independent experiments performed in duplicate.

    Techniques Used: Western Blot, Staining, Incubation, Cell Culture, Negative Control

    7) Product Images from "The ecdysis triggering hormone system is essential for successful moulting of a major hemimetabolous pest insect, Schistocerca gregaria"

    Article Title: The ecdysis triggering hormone system is essential for successful moulting of a major hemimetabolous pest insect, Schistocerca gregaria

    Journal: Scientific Reports

    doi: 10.1038/srep46502

    Intracellular signalling in SchgrETHR -expressing CHO-PAM28 and HEK293T cells. Receptor activation is shown as relative (%) to the highest value (100%) after normalization to the maximum response. Data represent the mean (±SEM) of three independent measurements, each performed in triplicate. ( A ) Dose-response curves for bioluminescence induced by Schgr ETH1 and Schgr ETH2 in SchgrETHR -expressing CHO-PAM28 cells. Zero response level is measured in cells challenged with BSA only. ( B ) Dose-response curves for luciferase bioluminescence induced by Schgr ETH1 and Schgr ETH2 in SchgrETHR -expressing HEK293T cells. Zero response level is measured in cells challenged with DMEM/IBMX.
    Figure Legend Snippet: Intracellular signalling in SchgrETHR -expressing CHO-PAM28 and HEK293T cells. Receptor activation is shown as relative (%) to the highest value (100%) after normalization to the maximum response. Data represent the mean (±SEM) of three independent measurements, each performed in triplicate. ( A ) Dose-response curves for bioluminescence induced by Schgr ETH1 and Schgr ETH2 in SchgrETHR -expressing CHO-PAM28 cells. Zero response level is measured in cells challenged with BSA only. ( B ) Dose-response curves for luciferase bioluminescence induced by Schgr ETH1 and Schgr ETH2 in SchgrETHR -expressing HEK293T cells. Zero response level is measured in cells challenged with DMEM/IBMX.

    Techniques Used: Expressing, Activation Assay, Luciferase

    8) Product Images from "Luciferase Reporter Gene Assay on Human, Murine and Rat Histamine H4 Receptor Orthologs: Correlations and Discrepancies between Distal and Proximal Readouts"

    Article Title: Luciferase Reporter Gene Assay on Human, Murine and Rat Histamine H4 Receptor Orthologs: Correlations and Discrepancies between Distal and Proximal Readouts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073961

    Effect of histamine and thioperamide on the luciferase activity in hH 4 R expressing cells. Concentration-response curves of histamine (HA) and thioperamide (THIO) on HEK293T-SF-hH 4 R-His 6 -CRE-Luc cells, stably co-expressing the CRE-controlled luciferase and the hH 4 R. The cells were pre-stimulated with 500 nM of forskolin alone or in combination with IBMX (50 µM). The effect of forskolin or that of forskolin plus IBMX was defined as 100% luciferase activity. Data points shown are the mean ± SEM of two independent experiments performed in triplicate.
    Figure Legend Snippet: Effect of histamine and thioperamide on the luciferase activity in hH 4 R expressing cells. Concentration-response curves of histamine (HA) and thioperamide (THIO) on HEK293T-SF-hH 4 R-His 6 -CRE-Luc cells, stably co-expressing the CRE-controlled luciferase and the hH 4 R. The cells were pre-stimulated with 500 nM of forskolin alone or in combination with IBMX (50 µM). The effect of forskolin or that of forskolin plus IBMX was defined as 100% luciferase activity. Data points shown are the mean ± SEM of two independent experiments performed in triplicate.

    Techniques Used: Luciferase, Activity Assay, Expressing, Concentration Assay, Stable Transfection

    9) Product Images from "Real-Time Imaging Reveals Augmentation of Glutamate-Induced Ca2+ Transients by the NO-cGMP Pathway in Cerebellar Granule Neurons"

    Article Title: Real-Time Imaging Reveals Augmentation of Glutamate-Induced Ca2+ Transients by the NO-cGMP Pathway in Cerebellar Granule Neurons

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19082185

    Real-time cGMP imaging in CGNs reveals degradation of cGMP by Zaprinast-sensitive PDEs. ( A ) cGi500-expressing CGNs isolated from R26-CAG-cGi500(L1) mice were stimulated three times with DEA/NO (50 nM), first with DEA/NO alone, then in the presence of IBMX (100 µM), and finally again with DEA/NO alone. The bar graph shows the statistical evaluation of DEA/NO-induced cGMP peak areas before (first peak), during (second peak), and after (third peak) incubation with IBMX. Peak areas were normalized to the first peak of each experiment. Data are presented as mean ± SEM; * p
    Figure Legend Snippet: Real-time cGMP imaging in CGNs reveals degradation of cGMP by Zaprinast-sensitive PDEs. ( A ) cGi500-expressing CGNs isolated from R26-CAG-cGi500(L1) mice were stimulated three times with DEA/NO (50 nM), first with DEA/NO alone, then in the presence of IBMX (100 µM), and finally again with DEA/NO alone. The bar graph shows the statistical evaluation of DEA/NO-induced cGMP peak areas before (first peak), during (second peak), and after (third peak) incubation with IBMX. Peak areas were normalized to the first peak of each experiment. Data are presented as mean ± SEM; * p

    Techniques Used: Imaging, Expressing, Isolation, Mouse Assay, Incubation

    10) Product Images from "Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK"

    Article Title: Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-17-56

    Adding FN or LAM during differentiation increases insulin release in response to elevated glucose concentration . MSCs were induced by four-stage protocol, and ELISA analysis for insulin release was performed for stage IV cells. (A) Insulin release at different glucose concentrations. Insulin release before and after treatment with (B) IBMX or (C) nifedipine. (mean ± S.D.; * P
    Figure Legend Snippet: Adding FN or LAM during differentiation increases insulin release in response to elevated glucose concentration . MSCs were induced by four-stage protocol, and ELISA analysis for insulin release was performed for stage IV cells. (A) Insulin release at different glucose concentrations. Insulin release before and after treatment with (B) IBMX or (C) nifedipine. (mean ± S.D.; * P

    Techniques Used: Laser Capture Microdissection, Concentration Assay, Enzyme-linked Immunosorbent Assay

    11) Product Images from "Collecting duct-specific knockout of adenylyl cyclase type VI causes a urinary concentration defect in mice"

    Article Title: Collecting duct-specific knockout of adenylyl cyclase type VI causes a urinary concentration defect in mice

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00397.2011

    AVP stimulated cAMP accumulation in acutely isolated inner medullary collecting duct (IMCD) from control and CD AC6 KO mice. IMCD were stimulated with 10 −9 M AVP in the presence of IBMX for 10 min. cAMP levels were assayed via ELISA and adjusted
    Figure Legend Snippet: AVP stimulated cAMP accumulation in acutely isolated inner medullary collecting duct (IMCD) from control and CD AC6 KO mice. IMCD were stimulated with 10 −9 M AVP in the presence of IBMX for 10 min. cAMP levels were assayed via ELISA and adjusted

    Techniques Used: Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay

    12) Product Images from "Signaling Properties and Pharmacological Analysis of Two Sulfakinin Receptors from the Red Flour Beetle, Tribolium castaneum"

    Article Title: Signaling Properties and Pharmacological Analysis of Two Sulfakinin Receptors from the Red Flour Beetle, Tribolium castaneum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094502

    Dose response curves for bioluminescence induced by s Trica -SK(5–13) in Trica -SKR expressing HEK293T cells. Luciferase bioluminescence induced in HEK293T cells transfected with Trica -SKR1 (A) or Trica -SKR2 (B) and CRE-luciferase construct. Receptor activation shown as the percentage of activation achieved with 100 nM sTrica -SK(5–13) (maximal response level = 100%). The zero response level corresponds to treatment of cells with DMEM/IBMX. Data represent the mean ± SEM of six (SKR1) or three (SKR2) independent measurements (each performed in duplicate).
    Figure Legend Snippet: Dose response curves for bioluminescence induced by s Trica -SK(5–13) in Trica -SKR expressing HEK293T cells. Luciferase bioluminescence induced in HEK293T cells transfected with Trica -SKR1 (A) or Trica -SKR2 (B) and CRE-luciferase construct. Receptor activation shown as the percentage of activation achieved with 100 nM sTrica -SK(5–13) (maximal response level = 100%). The zero response level corresponds to treatment of cells with DMEM/IBMX. Data represent the mean ± SEM of six (SKR1) or three (SKR2) independent measurements (each performed in duplicate).

    Techniques Used: Expressing, Luciferase, Transfection, Construct, Activation Assay

    13) Product Images from "Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand"

    Article Title: Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00969.2012

    β-AR stimulation by isoproterenol (ISO, 1 μM, n = 9), PDE inhibition by IBMX (100 μM, n = 9), protein phosphatase inhibition by calyculin A (1 μM, n = 10), or an increase in cAMP level by the membrane-permeable cAMP analog
    Figure Legend Snippet: β-AR stimulation by isoproterenol (ISO, 1 μM, n = 9), PDE inhibition by IBMX (100 μM, n = 9), protein phosphatase inhibition by calyculin A (1 μM, n = 10), or an increase in cAMP level by the membrane-permeable cAMP analog

    Techniques Used: Inhibition

    A : the effects of β-AR stimulation by ISO (1 μM, n = 5), PDE inhibition by IBMX (100 μM, n = 5), or protein phosphatase inhibition by calyculin A (1 μM, n = 5), or by the membrane-permeable cAMP analog CPT-cAMP (300 μM,
    Figure Legend Snippet: A : the effects of β-AR stimulation by ISO (1 μM, n = 5), PDE inhibition by IBMX (100 μM, n = 5), or protein phosphatase inhibition by calyculin A (1 μM, n = 5), or by the membrane-permeable cAMP analog CPT-cAMP (300 μM,

    Techniques Used: Inhibition, Cycling Probe Technology

    A : the effects of β-AR stimulation by ISO (1 μM, n = 5), PDE inhibition by IBMX (100 μM, n = 5), protein phosphatase inhibition by calyculin A (1 μM, n = 5), or by an increase in cAMP level by the membrane-permeable cAMP
    Figure Legend Snippet: A : the effects of β-AR stimulation by ISO (1 μM, n = 5), PDE inhibition by IBMX (100 μM, n = 5), protein phosphatase inhibition by calyculin A (1 μM, n = 5), or by an increase in cAMP level by the membrane-permeable cAMP

    Techniques Used: Inhibition

    The effects of β-AR stimulation by ISO (1 μM, n = 5), PDE inhibition by IBMX (100 μM, n = 5), or protein phosphatase inhibition by calyculin A (1 μM, n = 5), or by the membrane-permeable cAMP analog CPT-cAMP (300 μM,
    Figure Legend Snippet: The effects of β-AR stimulation by ISO (1 μM, n = 5), PDE inhibition by IBMX (100 μM, n = 5), or protein phosphatase inhibition by calyculin A (1 μM, n = 5), or by the membrane-permeable cAMP analog CPT-cAMP (300 μM,

    Techniques Used: Inhibition, Cycling Probe Technology

    14) Product Images from "H5N1 Virus Hemagglutinin Inhibition of cAMP-Dependent CFTR via TLR4-Mediated Janus Tyrosine Kinase 3 Activation Exacerbates Lung Inflammation"

    Article Title: H5N1 Virus Hemagglutinin Inhibition of cAMP-Dependent CFTR via TLR4-Mediated Janus Tyrosine Kinase 3 Activation Exacerbates Lung Inflammation

    Journal: Molecular Medicine

    doi: 10.2119/molmed.2014.00189

    HA-induced JAK3 activation stimulates Gαimediated inhibition of AC, resulting in a decrease in the intracellular cAMP level 16HBE cells were pretreated with IBMX (1 mmol/L )for 15 min and then treated with forskolin (FSK, 10 μmol/L, 30
    Figure Legend Snippet: HA-induced JAK3 activation stimulates Gαimediated inhibition of AC, resulting in a decrease in the intracellular cAMP level 16HBE cells were pretreated with IBMX (1 mmol/L )for 15 min and then treated with forskolin (FSK, 10 μmol/L, 30

    Techniques Used: Activation Assay, Inhibition

    15) Product Images from "Olfactory Signal Transduction in the Mouse Septal Organ"

    Article Title: Olfactory Signal Transduction in the Mouse Septal Organ

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi:

    The cAMP pathway mediates olfactory signal transduction in the septal organ. Inward currents were induced under voltage-clamp mode by different stimuli: A1 , the odorant mixture at 100 μ M; B1 , 10 μ M forskolin, an adenylyl cyclase activator; C1 , 300 μ M IBMX, a phosphodiesterase inhibitor; and D1 , 500 μ M 8-Br-cGMP, an activator of the CNG channel. The effects of 50 μ M MDL12330A, an adenylyl cyclase blocker on the responses induced by the following: A2 , the odorant mixture at 100 μ M; B2 , 10 μ M forskolin; C2 , 300 μ M IBMX; and D2 , 500 μ M 8-Br-cGMP. Recordings in the same row were from the same cell. The holding potentials were −50 mV for all recordings. E , Summary of responses under different conditions. Data are expressed as means ± SD and pooled from n cells, indicated as a number under each bar . The averaged responses induced by odorants, forskolin, and IBMX with MDL12330A are significantly smaller than those under control condition ( p
    Figure Legend Snippet: The cAMP pathway mediates olfactory signal transduction in the septal organ. Inward currents were induced under voltage-clamp mode by different stimuli: A1 , the odorant mixture at 100 μ M; B1 , 10 μ M forskolin, an adenylyl cyclase activator; C1 , 300 μ M IBMX, a phosphodiesterase inhibitor; and D1 , 500 μ M 8-Br-cGMP, an activator of the CNG channel. The effects of 50 μ M MDL12330A, an adenylyl cyclase blocker on the responses induced by the following: A2 , the odorant mixture at 100 μ M; B2 , 10 μ M forskolin; C2 , 300 μ M IBMX; and D2 , 500 μ M 8-Br-cGMP. Recordings in the same row were from the same cell. The holding potentials were −50 mV for all recordings. E , Summary of responses under different conditions. Data are expressed as means ± SD and pooled from n cells, indicated as a number under each bar . The averaged responses induced by odorants, forskolin, and IBMX with MDL12330A are significantly smaller than those under control condition ( p

    Techniques Used: Transduction

    16) Product Images from "Myosin1D is an evolutionarily conserved regulator of animal left–right asymmetry"

    Article Title: Myosin1D is an evolutionarily conserved regulator of animal left–right asymmetry

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04284-8

    myo1D controls the morphogenesis of the zebrafish left–right Organizer. a – e Compared to WT ( n = 104), MZ myo1D mutants ( n = 118) present a reduced KV size. Treatment with IBMX and Forskolin promotes KV lumen inflation and increases organ size in WT ( n = 66) and MZ myo1D mutants ( n = 85). e Dot plots of KV equatorial surface area in individual embryos. KV size is similar in embryos with normal or defective laterality. f IBMX/Forskolin treatment restores KV size ( d , e ), but not laterality in MZ myo1D mutants. g – j Cilia number and size are reduced in MZ myo1D mutants. g , h Projection of images from confocal stacks used to quantify number and length of cilia (acetylated tubulin, magenta) in the KV (ZO-1 positive cells, green). i , j Number and average length of cilia in individual embryos are reduced in MZ myo1D ( n = 117 embryos/4939 cilia) compared to WT ( n = 141/6967). k – m Confocal imaging of Arl13b-GFP labeled cilia in living embryos was used to analyze the number ( k ), motility ( l ) and positioning ( m ) of KV cilia. k Dot plot representing motile cilia number in individual embryos. MZ myo1D ( n = 42) mutants display lower motile cilia numbers compared to WT ( n = 33). Motile cilia numbers are however similar in MZ myo1D mutant embryos with normal or defective laterality. Total cilia numbers for this experiment are displayed in Supplementary Fig. 5 . l myo1D loss of function does not impair ciliary motility. m WT and MZ myo1D mutant animals display a similar enrichment of cilia in the anterior KV half. a – d , g , h are dorsal views of the KV, anterior up. All data collected at the eight-somites stage. Horizontal bars in e , i , j , k represent mean values. Error bars in l , m represent SEM. Scale bars: 20 µm in a – d ; 10 µm in g – h
    Figure Legend Snippet: myo1D controls the morphogenesis of the zebrafish left–right Organizer. a – e Compared to WT ( n = 104), MZ myo1D mutants ( n = 118) present a reduced KV size. Treatment with IBMX and Forskolin promotes KV lumen inflation and increases organ size in WT ( n = 66) and MZ myo1D mutants ( n = 85). e Dot plots of KV equatorial surface area in individual embryos. KV size is similar in embryos with normal or defective laterality. f IBMX/Forskolin treatment restores KV size ( d , e ), but not laterality in MZ myo1D mutants. g – j Cilia number and size are reduced in MZ myo1D mutants. g , h Projection of images from confocal stacks used to quantify number and length of cilia (acetylated tubulin, magenta) in the KV (ZO-1 positive cells, green). i , j Number and average length of cilia in individual embryos are reduced in MZ myo1D ( n = 117 embryos/4939 cilia) compared to WT ( n = 141/6967). k – m Confocal imaging of Arl13b-GFP labeled cilia in living embryos was used to analyze the number ( k ), motility ( l ) and positioning ( m ) of KV cilia. k Dot plot representing motile cilia number in individual embryos. MZ myo1D ( n = 42) mutants display lower motile cilia numbers compared to WT ( n = 33). Motile cilia numbers are however similar in MZ myo1D mutant embryos with normal or defective laterality. Total cilia numbers for this experiment are displayed in Supplementary Fig. 5 . l myo1D loss of function does not impair ciliary motility. m WT and MZ myo1D mutant animals display a similar enrichment of cilia in the anterior KV half. a – d , g , h are dorsal views of the KV, anterior up. All data collected at the eight-somites stage. Horizontal bars in e , i , j , k represent mean values. Error bars in l , m represent SEM. Scale bars: 20 µm in a – d ; 10 µm in g – h

    Techniques Used: Imaging, Labeling, Mutagenesis

    17) Product Images from "PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases"

    Article Title: PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200605050

    The cAMP response to PGE 1 is higher under the plasma membrane than in the bulk cytosol. (A) Wide-field images of a representative HEK293 cell cotransfected with R-CFP (top left) and C-YFP (not depicted) and with mp R-CFP (bottom left) and C-YFP (not depicted). For the same cell, the pseudocolor images of the 480/545-nm emission ratio before (time = 0 s) and after the addition of 10 μM PGE 1 (time = 200 s) and 100 μM IBMX (time = 800 s) are shown on the right. Bars, 10 μm. (B) Kinetics of cAMP changes recorded in the cells shown in A. Open circles represent kinetics recorded in the bulk cytosol with PKA-GFP, and closed circles represent kinetics recorded at the plasma membrane with mp PKA-GFP. (C) Summary of all the experiments performed in the same conditions as in A and B. (D) Time to reach half-maximal response (t/2) to 10 μM PGE 1 . (E) The summary of experiments performed by applying 1 μM PGE 1 . Error bars indicate SEM. *, 0.01
    Figure Legend Snippet: The cAMP response to PGE 1 is higher under the plasma membrane than in the bulk cytosol. (A) Wide-field images of a representative HEK293 cell cotransfected with R-CFP (top left) and C-YFP (not depicted) and with mp R-CFP (bottom left) and C-YFP (not depicted). For the same cell, the pseudocolor images of the 480/545-nm emission ratio before (time = 0 s) and after the addition of 10 μM PGE 1 (time = 200 s) and 100 μM IBMX (time = 800 s) are shown on the right. Bars, 10 μm. (B) Kinetics of cAMP changes recorded in the cells shown in A. Open circles represent kinetics recorded in the bulk cytosol with PKA-GFP, and closed circles represent kinetics recorded at the plasma membrane with mp PKA-GFP. (C) Summary of all the experiments performed in the same conditions as in A and B. (D) Time to reach half-maximal response (t/2) to 10 μM PGE 1 . (E) The summary of experiments performed by applying 1 μM PGE 1 . Error bars indicate SEM. *, 0.01

    Techniques Used:

    A unimolecular Epac-based sensor detects different [cAMP] at the plasma membrane and in the bulk cytosol. (A) Schematic representation of the fusion protein constituting the Epac-based cAMP sensors H30 and confocal micrographs showing its distribution in HEK293 cells. (B) Structure and localization of the membrane-targeted version of mp H30. Bars, 10 μm. (C) cAMP dose-response curves measured as the percent FRET changes of H30 (white circles), mp H30 (black circles), and nls H30 (gray circles). EC 50 are 12.5, 20, and 17.5 μM, respectively. (D) Representative kinetics of cAMP changes generated in the cytosol (white circles) and at the plasma membrane (black circles) upon stimulation with 1 μM PGE 1 followed by 100 μM IBMX. (E) Summary of the experiments performed as in D. Error bars represent SEM. **, P = 0.002; ***, P = 10 −4 .
    Figure Legend Snippet: A unimolecular Epac-based sensor detects different [cAMP] at the plasma membrane and in the bulk cytosol. (A) Schematic representation of the fusion protein constituting the Epac-based cAMP sensors H30 and confocal micrographs showing its distribution in HEK293 cells. (B) Structure and localization of the membrane-targeted version of mp H30. Bars, 10 μm. (C) cAMP dose-response curves measured as the percent FRET changes of H30 (white circles), mp H30 (black circles), and nls H30 (gray circles). EC 50 are 12.5, 20, and 17.5 μM, respectively. (D) Representative kinetics of cAMP changes generated in the cytosol (white circles) and at the plasma membrane (black circles) upon stimulation with 1 μM PGE 1 followed by 100 μM IBMX. (E) Summary of the experiments performed as in D. Error bars represent SEM. **, P = 0.002; ***, P = 10 −4 .

    Techniques Used: Generated

    Role of PKA in shaping the cAMP gradient between the plasma membrane and the cytosol. (A and B) Representative cAMP kinetic ( Romoser et al., 1996 ) and summary of experiments (B) performed in HEK293 cells cotransfected with PKA and either H30 or mp H30 and challenged with 1 μM PGE 1 followed by either total PDE inhibition with 100 μM IBMX or PKA inhibition with 10 μM H89 as indicated. (C and D) Representative kinetics (C) and summary of experiments (D) showing the effect of endogenous PKA inhibition on the cAMP response induced by 10 nM PGE 1 at the plasma membrane and bulk cytosol in the absence and presence of the PKA inhibitor H89 (10 μM). In all of the experiments, when the PKA inhibitor was used, cells were preincubated for 10 min with H89, and the inhibitor was present throughout the experiment. Error bars represent SEM. *, P = 0.02; **, P = 0.009; ***, P = 0.0009.
    Figure Legend Snippet: Role of PKA in shaping the cAMP gradient between the plasma membrane and the cytosol. (A and B) Representative cAMP kinetic ( Romoser et al., 1996 ) and summary of experiments (B) performed in HEK293 cells cotransfected with PKA and either H30 or mp H30 and challenged with 1 μM PGE 1 followed by either total PDE inhibition with 100 μM IBMX or PKA inhibition with 10 μM H89 as indicated. (C and D) Representative kinetics (C) and summary of experiments (D) showing the effect of endogenous PKA inhibition on the cAMP response induced by 10 nM PGE 1 at the plasma membrane and bulk cytosol in the absence and presence of the PKA inhibitor H89 (10 μM). In all of the experiments, when the PKA inhibitor was used, cells were preincubated for 10 min with H89, and the inhibitor was present throughout the experiment. Error bars represent SEM. *, P = 0.02; **, P = 0.009; ***, P = 0.0009.

    Techniques Used: Inhibition

    18) Product Images from "Changes in cGMP Levels Affect the Localization of EGL-4 in AWC in Caenorhabditis elegans"

    Article Title: Changes in cGMP Levels Affect the Localization of EGL-4 in AWC in Caenorhabditis elegans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031614

    Decreases in cGMP levels direct the nuclear entry of GFP::EGL-4 in AWC and promote adaptation. (A) Percent of animals displaying nuclear GFP::EGL-4 in AWC of naive (black bars), benzaldehyde-exposed (gray bars) and butanone-exposed (white bars) treatments. **Indicates p ≤0.005 significant differences between nuclear GFP::EGL-4 values of wildtype unadapted animals and odr-1 or daf-11 mutant unadapted animals, and also significant differences between wildtype adapted and pde quadruple mutant adapted values. (B) Chemotaxis response of PDE mutants and the guanylyl cyclase mutants daf-11 and odr-1 to the AWC sensed odor benzaldehyde. “−” indicates unexposed animals and “+” indicates exposed animals. **Indicates p ≤0.005 significant differences between chemotaxis index (CI) values between wildtype unexposed animals and mutant unexposed animals. *Indicates p ≤0.05 significant differences between wildtype odor-exposed CI values and mutant odor-exposed CI values. (C) Populations of GFP::EGL-4 ( pyIs500 ) expressing animals were exposed to the odor benzaldehyde with or without the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Populations exposed to 10 mM concentrations of IBMX displayed a reduced number of animals exhibiting nuclear GFP::EGL-4 in AWC at 80 minutes post exposure to benzaldehyde and IBMX. *Indicates p ≤0.05 significant differences between odor treated animals with IBMX versus odor treated animals without IBMX. (D) Expression of the cGMP phosphodiesterase PDE-3 under a heat-inducible promoter causes some increase in the number of animals displaying nuclear GFP::EGL-4. *Indicates p ≤0.05 significant differences between wildtype and transgenic animals after heat induction; **Indicates p ≤0.005 significant differences between transgenic animals with and without heat induction. “−” indicates no heat induction and “+” indicates after heat induction. P values calculated using the Student's t -test. Error bars represent the S.E.M.
    Figure Legend Snippet: Decreases in cGMP levels direct the nuclear entry of GFP::EGL-4 in AWC and promote adaptation. (A) Percent of animals displaying nuclear GFP::EGL-4 in AWC of naive (black bars), benzaldehyde-exposed (gray bars) and butanone-exposed (white bars) treatments. **Indicates p ≤0.005 significant differences between nuclear GFP::EGL-4 values of wildtype unadapted animals and odr-1 or daf-11 mutant unadapted animals, and also significant differences between wildtype adapted and pde quadruple mutant adapted values. (B) Chemotaxis response of PDE mutants and the guanylyl cyclase mutants daf-11 and odr-1 to the AWC sensed odor benzaldehyde. “−” indicates unexposed animals and “+” indicates exposed animals. **Indicates p ≤0.005 significant differences between chemotaxis index (CI) values between wildtype unexposed animals and mutant unexposed animals. *Indicates p ≤0.05 significant differences between wildtype odor-exposed CI values and mutant odor-exposed CI values. (C) Populations of GFP::EGL-4 ( pyIs500 ) expressing animals were exposed to the odor benzaldehyde with or without the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Populations exposed to 10 mM concentrations of IBMX displayed a reduced number of animals exhibiting nuclear GFP::EGL-4 in AWC at 80 minutes post exposure to benzaldehyde and IBMX. *Indicates p ≤0.05 significant differences between odor treated animals with IBMX versus odor treated animals without IBMX. (D) Expression of the cGMP phosphodiesterase PDE-3 under a heat-inducible promoter causes some increase in the number of animals displaying nuclear GFP::EGL-4. *Indicates p ≤0.05 significant differences between wildtype and transgenic animals after heat induction; **Indicates p ≤0.005 significant differences between transgenic animals with and without heat induction. “−” indicates no heat induction and “+” indicates after heat induction. P values calculated using the Student's t -test. Error bars represent the S.E.M.

    Techniques Used: Mutagenesis, Chemotaxis Assay, Expressing, Transgenic Assay

    19) Product Images from "Interferon-? inhibits ghrelin expression and secretion via a somatostatin-mediated mechanism"

    Article Title: Interferon-? inhibits ghrelin expression and secretion via a somatostatin-mediated mechanism

    Journal: World Journal of Gastroenterology : WJG

    doi: 10.3748/wjg.v17.i26.3117

    The somatostatin receptor agonist octreotide inhibits activation of the ghrelin promoter. Pulmonary carcinoid NCI-H727 cells were transfected with a 2 kb mouse ghrelin promoter construct and treated for 24 h with forskolin, IBMX, octreotide, and interferon γ (IFNγ) alone or in combination, after which the ghrelin promoter response was measured by the dual-luciferase reporter assay system. Octreotide inhibited both basal and IBMX and/or forskolin-induced ghrelin expression. Forskolin and IBMX were dissolved in dimethyl sulfoxide (DMSO). a P
    Figure Legend Snippet: The somatostatin receptor agonist octreotide inhibits activation of the ghrelin promoter. Pulmonary carcinoid NCI-H727 cells were transfected with a 2 kb mouse ghrelin promoter construct and treated for 24 h with forskolin, IBMX, octreotide, and interferon γ (IFNγ) alone or in combination, after which the ghrelin promoter response was measured by the dual-luciferase reporter assay system. Octreotide inhibited both basal and IBMX and/or forskolin-induced ghrelin expression. Forskolin and IBMX were dissolved in dimethyl sulfoxide (DMSO). a P

    Techniques Used: Activation Assay, Transfection, Construct, Luciferase, Reporter Assay, Expressing

    20) Product Images from "Chromatin Accessibility and Transcription Factor Binding at the PPARγ2 Promoter during Adipogenesis is Protein Kinase A-Dependent"

    Article Title: Chromatin Accessibility and Transcription Factor Binding at the PPARγ2 Promoter during Adipogenesis is Protein Kinase A-Dependent

    Journal: Journal of Cellular Physiology

    doi: 10.1002/jcp.22308

    Chromatin accessibility changes at the PPARγ2 promoter are dependent upon increased cAMP levels. 3T3-L1 cells were differentiated for 3 hours in the presence of DMEM containing 10% fetal calf serum plus insulin alone (I), dexamethasone alone (D), IBMX alone (M), all three (MDI) or no additional additives (−) and subsequently analyzed by (A) Southern blot or (B) LM-PCR for Stu I accessibility at the HS1 site on the PPARγ2 promoter. (C, D) 3T3-L1 cells were differentiated for 3 hours in the presence of DMEM containing 10% fetal calf serum (−) or DMEM containing 10% fetal calf serum plus dibutyryl-cAMP (db-cAMP) and assayed for Stu I accessibility changes at the HS1 site on the PPARγ2 promoter by Southern blot (C) or LM-PCR (D).
    Figure Legend Snippet: Chromatin accessibility changes at the PPARγ2 promoter are dependent upon increased cAMP levels. 3T3-L1 cells were differentiated for 3 hours in the presence of DMEM containing 10% fetal calf serum plus insulin alone (I), dexamethasone alone (D), IBMX alone (M), all three (MDI) or no additional additives (−) and subsequently analyzed by (A) Southern blot or (B) LM-PCR for Stu I accessibility at the HS1 site on the PPARγ2 promoter. (C, D) 3T3-L1 cells were differentiated for 3 hours in the presence of DMEM containing 10% fetal calf serum (−) or DMEM containing 10% fetal calf serum plus dibutyryl-cAMP (db-cAMP) and assayed for Stu I accessibility changes at the HS1 site on the PPARγ2 promoter by Southern blot (C) or LM-PCR (D).

    Techniques Used: Southern Blot, Polymerase Chain Reaction

    Binding of C/EBPβ and c-Fos to the PPARγ2 promoter is dependent upon the PKA signaling pathway. 3T3-L1 cells were differentiated for the indicated number of hours in the presence of complete differentiation media and harvested for ChIP assays. (A) Binding of C/EBPβ and c-Fos to the PPARγ2 promoter. (B, C) 3T3-L1 cells were differentiated for 3 hours in DMEM containing 10% fetal calf serum alone (−), or with insulin (I), dexamethasone (D), IBMX (M), forskalin, db-cAMP, or complete differentiation media (MDI) and harvested for ChIP assays to assess C/EBPβ binding (B) or c-Fos binding (C) to the PPARγ2 promoter. (D) 2 day post-confluent 3T3-L1 cells were treated with the indicated compounds for 1 hour prior to the addition of complete differentiation media for 3 hours, then harvested for ChIP assays to assess the binding of C/EBPβ (left) or c-Fos (right) to the PPARγ2 promoter.
    Figure Legend Snippet: Binding of C/EBPβ and c-Fos to the PPARγ2 promoter is dependent upon the PKA signaling pathway. 3T3-L1 cells were differentiated for the indicated number of hours in the presence of complete differentiation media and harvested for ChIP assays. (A) Binding of C/EBPβ and c-Fos to the PPARγ2 promoter. (B, C) 3T3-L1 cells were differentiated for 3 hours in DMEM containing 10% fetal calf serum alone (−), or with insulin (I), dexamethasone (D), IBMX (M), forskalin, db-cAMP, or complete differentiation media (MDI) and harvested for ChIP assays to assess C/EBPβ binding (B) or c-Fos binding (C) to the PPARγ2 promoter. (D) 2 day post-confluent 3T3-L1 cells were treated with the indicated compounds for 1 hour prior to the addition of complete differentiation media for 3 hours, then harvested for ChIP assays to assess the binding of C/EBPβ (left) or c-Fos (right) to the PPARγ2 promoter.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    21) Product Images from "Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction"

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

    Journal: Adipocyte

    doi: 10.1080/21623945.2020.1720480

    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
    Figure Legend 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

    Techniques Used:

    22) Product Images from "Transfer, analysis and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors"

    Article Title: Transfer, analysis and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    doi: 10.1359/jbmr.091036

    Lentiviral transfer of Gsα R201C mutation in hBMSCs (A) LV vectors used for Gsα R201C transfer. LV-EF-Gsα R201C encodes the Gsα R201C protein (HA-tagged), controlled by the EF-1α promoter. The bidirectional LV vector (LV-BD-Gsα R201C ) encodes Gsα R201C and eGFP, under the control of the PGK and the CMV minimal promoters respectively. (B) Western blot analysis of Gsα R201C expression in LV-EF-Gsα R201C -transduced hBMSCs. Immunoblotting for HA demonstrates the specific signal for the mutated Gsα protein. Immunoblotting for Gsα demonstrates that mock-treated and empty vector (LV-Ctr)-transduced cells express comparable amounts of the 48KD and 43KD isoforms of Gsα (Gsα- long and Gsα- short , respectively) resulting from alternative splicing of exon 3. A specific increase of Gsα- long is observed in cells transduced with LV-EF-Gsα R201C . (C) Gsα R201C expression in hBMSCs is detected by immunofluorescence, as indicated by HA immunolabeling. (D) Intracellular cAMP levels were measured in control or LV-EF-Gsα R201C -transduced hBMSCs. In the presence of IBMX (1 mM) alone, or of IBMX and forskolin (10 μM), significantly higher levels of cAMP are observed in Gsα R201C expressing cells compared to control cells. Data from 5 separate experiments in duplicate are expressed as mean ± SD. a, p
    Figure Legend Snippet: Lentiviral transfer of Gsα R201C mutation in hBMSCs (A) LV vectors used for Gsα R201C transfer. LV-EF-Gsα R201C encodes the Gsα R201C protein (HA-tagged), controlled by the EF-1α promoter. The bidirectional LV vector (LV-BD-Gsα R201C ) encodes Gsα R201C and eGFP, under the control of the PGK and the CMV minimal promoters respectively. (B) Western blot analysis of Gsα R201C expression in LV-EF-Gsα R201C -transduced hBMSCs. Immunoblotting for HA demonstrates the specific signal for the mutated Gsα protein. Immunoblotting for Gsα demonstrates that mock-treated and empty vector (LV-Ctr)-transduced cells express comparable amounts of the 48KD and 43KD isoforms of Gsα (Gsα- long and Gsα- short , respectively) resulting from alternative splicing of exon 3. A specific increase of Gsα- long is observed in cells transduced with LV-EF-Gsα R201C . (C) Gsα R201C expression in hBMSCs is detected by immunofluorescence, as indicated by HA immunolabeling. (D) Intracellular cAMP levels were measured in control or LV-EF-Gsα R201C -transduced hBMSCs. In the presence of IBMX (1 mM) alone, or of IBMX and forskolin (10 μM), significantly higher levels of cAMP are observed in Gsα R201C expressing cells compared to control cells. Data from 5 separate experiments in duplicate are expressed as mean ± SD. a, p

    Techniques Used: Mutagenesis, Plasmid Preparation, Western Blot, Expressing, Transduction, Immunofluorescence, Immunolabeling

    Activity, mRNA expression and phenotypic effects of PDEs in mutation-transduced hBMSCs (A) PDE activity was assayed in hBMSCs, mock-treated or transduced with LV-Ctr or LV- EF-Gsα R201C , using 10 μM of cAMP as a substrate, in the presence or absence of either IBMX (1 mM) or Rolipram (20 μM). b, p
    Figure Legend Snippet: Activity, mRNA expression and phenotypic effects of PDEs in mutation-transduced hBMSCs (A) PDE activity was assayed in hBMSCs, mock-treated or transduced with LV-Ctr or LV- EF-Gsα R201C , using 10 μM of cAMP as a substrate, in the presence or absence of either IBMX (1 mM) or Rolipram (20 μM). b, p

    Techniques Used: Activity Assay, Expressing, Mutagenesis, Transduction

    23) Product Images from "The norpurpureine alkaloid from Annona purpurea inhibits human platelet activation in vitro"

    Article Title: The norpurpureine alkaloid from Annona purpurea inhibits human platelet activation in vitro

    Journal: Cellular & Molecular Biology Letters

    doi: 10.1186/s11658-018-0082-4

    Norpurpureine prevents the agonist-induced decrease in intra-platelet cAMP levels. Platelets were pre-treated for 10 min with vehicle (0.25% DMSO), 220 μM norpurpureine and 10 μM IBMX; and stimulated with 1 μg/ml collagen (in platelet-rich plasma, PRP) and 0.075 U/ml thrombin (in washed platelets, WP). Data are the means ± SD ( n = 2, done in triplicate). p
    Figure Legend Snippet: Norpurpureine prevents the agonist-induced decrease in intra-platelet cAMP levels. Platelets were pre-treated for 10 min with vehicle (0.25% DMSO), 220 μM norpurpureine and 10 μM IBMX; and stimulated with 1 μg/ml collagen (in platelet-rich plasma, PRP) and 0.075 U/ml thrombin (in washed platelets, WP). Data are the means ± SD ( n = 2, done in triplicate). p

    Techniques Used:

    24) Product Images from "A melanocyte lineage program confers resistance to MAP kinase pathway inhibition"

    Article Title: A melanocyte lineage program confers resistance to MAP kinase pathway inhibition

    Journal: Nature

    doi: 10.1038/nature12688

    A cyclic AMP signaling network mediates resistance to RAF, MEK and ERK inhibitors a, Average fold change (relative to DMSO) in MAPK-i GI 50 or AUC in a panel of BRAF V600 -mutant cell lines treated with vehicle (DMSO), forskolin and IBMX (FSK/I) or dbcAMP and IBMX (cAMP/I). n=8 technical replicates, representative of 3 independent experiments. b, Heat map showing relative cell viability (percent of DMSO) following treatment with forskolin and IBMX (FSK/I) or dbcAMP and IBMX (cAMP/I) in the presence of vehicle (DMSO), the PKA inhibitor H89 and a single dose of indicated MAPK-i. c, Immunoblot analysis of phosphorylated CREB/ATF1(Ser133/Ser63) in lysates from WM266.4 virally transduced with the indicated expression constructs. d, Viability of WM266.4 expressing either LacZ (control) or dominant-negative CREB alleles (CREB R301L or A-CREB) following treatment with forskolin and IBMX (FSK/I) in the presence indicated MAPK-i. Viability is expressed as a percentage of DMSO. Error bars represent s.d. of mean, n=6 technical replicates, representative of two independent experiments. e, Quantification of pCREB and pATF1 expression following immunoblot analysis of lysates extracted from BRAF V600 -mutant human tumors. Tumors were biopsied pre-initiation of treatment (P, n=5), following 10–14 days of MAPK-inhibitor treatment (on-treatment, O, n=6) or following relapse (R, n=7). MAPK-inhibitor therapy is noted. All available samples were tested and reported. Pre-treatment and on-treatment samples are paired. * p
    Figure Legend Snippet: A cyclic AMP signaling network mediates resistance to RAF, MEK and ERK inhibitors a, Average fold change (relative to DMSO) in MAPK-i GI 50 or AUC in a panel of BRAF V600 -mutant cell lines treated with vehicle (DMSO), forskolin and IBMX (FSK/I) or dbcAMP and IBMX (cAMP/I). n=8 technical replicates, representative of 3 independent experiments. b, Heat map showing relative cell viability (percent of DMSO) following treatment with forskolin and IBMX (FSK/I) or dbcAMP and IBMX (cAMP/I) in the presence of vehicle (DMSO), the PKA inhibitor H89 and a single dose of indicated MAPK-i. c, Immunoblot analysis of phosphorylated CREB/ATF1(Ser133/Ser63) in lysates from WM266.4 virally transduced with the indicated expression constructs. d, Viability of WM266.4 expressing either LacZ (control) or dominant-negative CREB alleles (CREB R301L or A-CREB) following treatment with forskolin and IBMX (FSK/I) in the presence indicated MAPK-i. Viability is expressed as a percentage of DMSO. Error bars represent s.d. of mean, n=6 technical replicates, representative of two independent experiments. e, Quantification of pCREB and pATF1 expression following immunoblot analysis of lysates extracted from BRAF V600 -mutant human tumors. Tumors were biopsied pre-initiation of treatment (P, n=5), following 10–14 days of MAPK-inhibitor treatment (on-treatment, O, n=6) or following relapse (R, n=7). MAPK-inhibitor therapy is noted. All available samples were tested and reported. Pre-treatment and on-treatment samples are paired. * p

    Techniques Used: Mutagenesis, Transduction, Expressing, Construct, Dominant Negative Mutation

    cAMP/PKA regulation of MITF mediates resistance to MAPK pathway inhibition a , Immunoblot analysis and b , quantification in lysates from WM266.4 cells treated as indicated. Arrow indicates the slower migrating, phosphorylated form of MITF, error bars represent s.d. of mean, n=3 biological replicates. c , Western blot analysis of WM266.4 following treatment with AZD6244 and stimulated for the indicated times with forskolin/IBMX. Forskolin/IBMX was washed out of the cells and replenished with normal growth media. Cell lysates were collected at the indicated times. d , Immunoblot analysis of WM266.4 cells following treatment with forskolin/IBMX (FSK/I) for the indicated times in the presence of vehicle (DMSO) or MEK-i. Genes identified in resistance screens are underlined. e , Immunoblot analysis of a panel of BRAF V600 -mutant malignant melanoma cell lines following treatment with AZD6244 in the presence of vehicle (DMSO), forskolin/IBMX (FSK/I) or dbcAMP/IBMX (cAMP/I). f , Immunoblot analysis of WM266.4 cells following treatment with forskolin/IBMX (FSK/I) in the presence of vehicle (DMSO) or indicated MAPK-pathway inhibitor. g , Gene signatures for all candidates and controls were generated in A375 and compared to the signatures of cAMP-stimulating small molecules, including forskolin and its water-soluble derivative, colforsin. Individual genes are grouped as Candidates or Neutral controls, with each gene represented by a vertical line. Genes are ranked by similarity with colforsin, with #1 being the most similar. A subset of the most similar genes is noted. h , Immunoblot analysis of WM266.4 after viral expression of the indicated genes or treatment with forskolin/IBMX (FSK/I) in the presence of vehicle (DMSO) or AZD6244.
    Figure Legend Snippet: cAMP/PKA regulation of MITF mediates resistance to MAPK pathway inhibition a , Immunoblot analysis and b , quantification in lysates from WM266.4 cells treated as indicated. Arrow indicates the slower migrating, phosphorylated form of MITF, error bars represent s.d. of mean, n=3 biological replicates. c , Western blot analysis of WM266.4 following treatment with AZD6244 and stimulated for the indicated times with forskolin/IBMX. Forskolin/IBMX was washed out of the cells and replenished with normal growth media. Cell lysates were collected at the indicated times. d , Immunoblot analysis of WM266.4 cells following treatment with forskolin/IBMX (FSK/I) for the indicated times in the presence of vehicle (DMSO) or MEK-i. Genes identified in resistance screens are underlined. e , Immunoblot analysis of a panel of BRAF V600 -mutant malignant melanoma cell lines following treatment with AZD6244 in the presence of vehicle (DMSO), forskolin/IBMX (FSK/I) or dbcAMP/IBMX (cAMP/I). f , Immunoblot analysis of WM266.4 cells following treatment with forskolin/IBMX (FSK/I) in the presence of vehicle (DMSO) or indicated MAPK-pathway inhibitor. g , Gene signatures for all candidates and controls were generated in A375 and compared to the signatures of cAMP-stimulating small molecules, including forskolin and its water-soluble derivative, colforsin. Individual genes are grouped as Candidates or Neutral controls, with each gene represented by a vertical line. Genes are ranked by similarity with colforsin, with #1 being the most similar. A subset of the most similar genes is noted. h , Immunoblot analysis of WM266.4 after viral expression of the indicated genes or treatment with forskolin/IBMX (FSK/I) in the presence of vehicle (DMSO) or AZD6244.

    Techniques Used: Inhibition, Western Blot, Mutagenesis, Generated, Expressing

    Candidate resistance genes are transcriptional effectors of the MAPK and cAMP-pathways a, Schematic outlining the identification of candidate resistance genes endogenously regulated by cAMP. b, Quantification of TBP -normalized mRNA levels using real-time quantitative PCR (relative to DMSO-treatment) following a time course of MEK-i treatment. Error bars represent s.d. of mean, n=3 technical replicates representative of 3 independent experiments. c, Quantification of TBP -normalized mRNA levels using real-time quantitative PCR (relative to DMSO-treatment) following treatment with forskolin and IBMX (FSK/I) for the indicated times in the presence of vehicle (DMSO) or MEK-i. Error bars represent s.d. of mean, n=3 technical replicates representative of 2 independent experiments. d, Immunoblot analysis of lysates from WM266.4 treated with DMSO or MEK-i followed by treatment with panobinostat, vorinostat or entinostat and subsequently stimulated with forskolin and IBMX (FSK/I). e, Cellular viability of WM266.4 treated with the indicated combinations of MAPK-i, HDAC-i and forskolin and IBMX (FSK/I). Cell viability is shown as a percent of DMSO in un-stimulated/non drug-treated cells. Error bars represent s.d. of mean, n=6 technical replicates representative of 2 independent experiments. f, A mechanistic model of GPCR-mediated resistance.
    Figure Legend Snippet: Candidate resistance genes are transcriptional effectors of the MAPK and cAMP-pathways a, Schematic outlining the identification of candidate resistance genes endogenously regulated by cAMP. b, Quantification of TBP -normalized mRNA levels using real-time quantitative PCR (relative to DMSO-treatment) following a time course of MEK-i treatment. Error bars represent s.d. of mean, n=3 technical replicates representative of 3 independent experiments. c, Quantification of TBP -normalized mRNA levels using real-time quantitative PCR (relative to DMSO-treatment) following treatment with forskolin and IBMX (FSK/I) for the indicated times in the presence of vehicle (DMSO) or MEK-i. Error bars represent s.d. of mean, n=3 technical replicates representative of 2 independent experiments. d, Immunoblot analysis of lysates from WM266.4 treated with DMSO or MEK-i followed by treatment with panobinostat, vorinostat or entinostat and subsequently stimulated with forskolin and IBMX (FSK/I). e, Cellular viability of WM266.4 treated with the indicated combinations of MAPK-i, HDAC-i and forskolin and IBMX (FSK/I). Cell viability is shown as a percent of DMSO in un-stimulated/non drug-treated cells. Error bars represent s.d. of mean, n=6 technical replicates representative of 2 independent experiments. f, A mechanistic model of GPCR-mediated resistance.

    Techniques Used: Real-time Polymerase Chain Reaction

    Inhibition of PKA or MITF impairs cAMP-mediated resistance to MAPK pathway inhibitors a , Cell viability of WM266.4 expressing a control shRNA (shLuciferase) or shRNAs targeting MITF treated with indicated MAPK-i and concomitant treatment with either DMSO or forskolin/IBMX (FSK/I). Error bars represent s.d. of mean, n=6 technical replicates, data is representative of two independent experiments. b , Western blot analysis of WM266.4 expressing the shRNA-constructs used in a. c , Western blot analysis of WM266.4 treated with AZD6244, followed by pre-treatment with DMSO or H89 and subsequent stimulation with forskolin/IBMX (FSK/I) for the indicated times. d , Immunoblot analysis of lysates extracted from human BRAF V600E positive melanoma biopsies. Biopsies were obtained prior to treatment (P), on MAPK-inhibitor treatment for 10–14 days (on-treatment, O) or following relapse (R). e , Immunoblot analysis of WM266.4 treated with the indicated concentration of HDAC-inhibitor. f , Immunoblot analysis of SKMEL19 and SKMEL28 in the presence of vehicle (DMSO) or AZD6244, followed by treatment with the indicated HDAC-inhibitor (panobinostat; Pan, vorinostat; Vor) and subsequent stimulation with forskolin/IBMX (FSK/I). g , Drug sensitivity curves of Panobinostat and Vorinostat in WM266.4 expressing LacZ or MITFm. Error bars represent s.d. of mean, n=3 technical replicates.
    Figure Legend Snippet: Inhibition of PKA or MITF impairs cAMP-mediated resistance to MAPK pathway inhibitors a , Cell viability of WM266.4 expressing a control shRNA (shLuciferase) or shRNAs targeting MITF treated with indicated MAPK-i and concomitant treatment with either DMSO or forskolin/IBMX (FSK/I). Error bars represent s.d. of mean, n=6 technical replicates, data is representative of two independent experiments. b , Western blot analysis of WM266.4 expressing the shRNA-constructs used in a. c , Western blot analysis of WM266.4 treated with AZD6244, followed by pre-treatment with DMSO or H89 and subsequent stimulation with forskolin/IBMX (FSK/I) for the indicated times. d , Immunoblot analysis of lysates extracted from human BRAF V600E positive melanoma biopsies. Biopsies were obtained prior to treatment (P), on MAPK-inhibitor treatment for 10–14 days (on-treatment, O) or following relapse (R). e , Immunoblot analysis of WM266.4 treated with the indicated concentration of HDAC-inhibitor. f , Immunoblot analysis of SKMEL19 and SKMEL28 in the presence of vehicle (DMSO) or AZD6244, followed by treatment with the indicated HDAC-inhibitor (panobinostat; Pan, vorinostat; Vor) and subsequent stimulation with forskolin/IBMX (FSK/I). g , Drug sensitivity curves of Panobinostat and Vorinostat in WM266.4 expressing LacZ or MITFm. Error bars represent s.d. of mean, n=3 technical replicates.

    Techniques Used: Inhibition, Expressing, shRNA, Western Blot, Construct, Concentration Assay

    Cyclic AMP induces CREB/ATF1 phosphorylation and induces MAPK-pathway inhibitor resistance a , Mean fold-change in intracellular cAMP following treatment with forskolin + IBMX (FSK/I) or dbcAMP + IBMX (cAMP/I) using a competitive cAMP ELISA assay (n=2 technical replicates, representative of 2 independent experiments). b , Bar graphs showing the change in the half-maximal inhibitory concentration (GI 50 ) of BRAF V600E -mutant cell lines treated with escalating doses of indicated MAPK-pathway inhibitor in the presence of vehicle (DMSO), forskolin/IBMX (FSK/IBMX) or dbcAMP/IBMX (cAMP/IBMX). c , Relative cell viability (percent of DMSO) following FSK/IBMX or cAMP/IBMX treatment in the absence of MAPK-pathway inhibitor treatment. Error bars represent s.d. of mean, n=8 technical replicates. Data is representative of 2 independent experiments. d , Number of viable cells treated with the indicated compounds in the presence of vehicle (DMSO) or forskolin and IBMX (FSK/I). Error bars represent s.d. of mean, n=3 technical replicates. e , Immunoblot analysis of WM983b following pre-treatment with the PKA inhibitor H89 and stimulation with forskolin and IBMX. f , Viability of WM266.4 treated with the indicated compounds and doses in the presence of vehicle (DMSO) or forskolin and IBMX (FSK/I). Error bars represent s.d. of mean, n=6 technical replicates.
    Figure Legend Snippet: Cyclic AMP induces CREB/ATF1 phosphorylation and induces MAPK-pathway inhibitor resistance a , Mean fold-change in intracellular cAMP following treatment with forskolin + IBMX (FSK/I) or dbcAMP + IBMX (cAMP/I) using a competitive cAMP ELISA assay (n=2 technical replicates, representative of 2 independent experiments). b , Bar graphs showing the change in the half-maximal inhibitory concentration (GI 50 ) of BRAF V600E -mutant cell lines treated with escalating doses of indicated MAPK-pathway inhibitor in the presence of vehicle (DMSO), forskolin/IBMX (FSK/IBMX) or dbcAMP/IBMX (cAMP/IBMX). c , Relative cell viability (percent of DMSO) following FSK/IBMX or cAMP/IBMX treatment in the absence of MAPK-pathway inhibitor treatment. Error bars represent s.d. of mean, n=8 technical replicates. Data is representative of 2 independent experiments. d , Number of viable cells treated with the indicated compounds in the presence of vehicle (DMSO) or forskolin and IBMX (FSK/I). Error bars represent s.d. of mean, n=3 technical replicates. e , Immunoblot analysis of WM983b following pre-treatment with the PKA inhibitor H89 and stimulation with forskolin and IBMX. f , Viability of WM266.4 treated with the indicated compounds and doses in the presence of vehicle (DMSO) or forskolin and IBMX (FSK/I). Error bars represent s.d. of mean, n=6 technical replicates.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Mutagenesis

    Candidate GPCR/PKA pathway genes induce cAMP and CREB/ATF1 phosphorylation a , Western blot of BRAF V600 -mutant melanoma cell lines stimulated with forskolin/IBMX or cAMP/IBMX. b , Western blot analysis of WM266.4 treated with AZD6244, followed by stimulation with forskolin and IBMX (FSK/IBMX). c , Western blot analysis of 293T lysates transfected with indicated genes or stimulated with forskolin/IBMX. d , Quantification of immunoblot analyses of 293T transiently transfected with the indicated expression constructs, pre-treated with IBMX (arbitrary units, n=2 biological replicates). e , Mean control or candidate gene-induced cAMP production was measured following transfection of 293T with indicated expression constructs or treatment with forskolin and IBMX (FSK/I). cAMP levels were determined using an immuno-competition assay in the presence (red bars) or absence (black bars) of IBMX (n=2 technical replicates, data is representative of three independent experiments). The green dashed line represents levels of cAMP in negative controls (eGFP, Luciferase, LacZ). f , Western blot analysis of WM266.4 expressing indicated constructs and treated with AZD6244 and/or forskolin/IBMX (FSK/IBMX).
    Figure Legend Snippet: Candidate GPCR/PKA pathway genes induce cAMP and CREB/ATF1 phosphorylation a , Western blot of BRAF V600 -mutant melanoma cell lines stimulated with forskolin/IBMX or cAMP/IBMX. b , Western blot analysis of WM266.4 treated with AZD6244, followed by stimulation with forskolin and IBMX (FSK/IBMX). c , Western blot analysis of 293T lysates transfected with indicated genes or stimulated with forskolin/IBMX. d , Quantification of immunoblot analyses of 293T transiently transfected with the indicated expression constructs, pre-treated with IBMX (arbitrary units, n=2 biological replicates). e , Mean control or candidate gene-induced cAMP production was measured following transfection of 293T with indicated expression constructs or treatment with forskolin and IBMX (FSK/I). cAMP levels were determined using an immuno-competition assay in the presence (red bars) or absence (black bars) of IBMX (n=2 technical replicates, data is representative of three independent experiments). The green dashed line represents levels of cAMP in negative controls (eGFP, Luciferase, LacZ). f , Western blot analysis of WM266.4 expressing indicated constructs and treated with AZD6244 and/or forskolin/IBMX (FSK/IBMX).

    Techniques Used: Western Blot, Mutagenesis, Transfection, Expressing, Construct, Competitive Binding Assay, Luciferase

    Broad validation of candidate resistance genes in a panel of BRAF V600 -mutant melanoma cell lines a , Drug sensitivity curves for PLX4720, AZD6244 and VRT11E in the panel of 8 BRAF V600E -mutant malignant melanoma cell lines used for the primary and validation screening experiments (described in Main Fig. 2). Error bars represent s.d. of mean, n=6 technical replicates. b , Western blot analysis following treatment with indicated MAPK-i in the panel of 8 BRAF V600E -mutant malignant melanoma cell lines used in a. c , Box plot of all candidate and control ORF infection efficiencies in the panel of 8 cell lines used in the validation screening experiments. Center line represents the median value, box defines the 25 th −75 th percentile and whiskers define the 5–95% confidence interval. Outliers are shown as individual data points. d , Summary of the cellular viability (relative to DMSO) of negative and neutral control genes observed in validation screens. Bar graph shows the average viability (relative to that of DMSO treatment) of each cell line when expressing the 59 negative and neutral control genes included in all validation screening experiments. Error bars represent s.d. of mean, each measured in technical duplicates. e , Average composite rescue score of each class of proteins identified among the resistance candidates (relates to Main Fig. 2 ). Number of genes within each protein class is shown in parentheses. f , ADCY9 was identified as a resistance candidate in the primary resistance screen, but was a DNA failure in our independent prep of candidate virus. Therefore, ADCY9 was not included in the high throughput validation screens, but was included in all subsequent validation work. These data show that ADCY9 is able to confer resistance to all tested MAPK-i to a similar degree as forskolin/IBMX treatment. Error bars represent s.d. of mean, n=6 technical replicates. g , Western blot analysis of the expression of V5-epitope tagged eGFP and ADCY9 in WM266.4.
    Figure Legend Snippet: Broad validation of candidate resistance genes in a panel of BRAF V600 -mutant melanoma cell lines a , Drug sensitivity curves for PLX4720, AZD6244 and VRT11E in the panel of 8 BRAF V600E -mutant malignant melanoma cell lines used for the primary and validation screening experiments (described in Main Fig. 2). Error bars represent s.d. of mean, n=6 technical replicates. b , Western blot analysis following treatment with indicated MAPK-i in the panel of 8 BRAF V600E -mutant malignant melanoma cell lines used in a. c , Box plot of all candidate and control ORF infection efficiencies in the panel of 8 cell lines used in the validation screening experiments. Center line represents the median value, box defines the 25 th −75 th percentile and whiskers define the 5–95% confidence interval. Outliers are shown as individual data points. d , Summary of the cellular viability (relative to DMSO) of negative and neutral control genes observed in validation screens. Bar graph shows the average viability (relative to that of DMSO treatment) of each cell line when expressing the 59 negative and neutral control genes included in all validation screening experiments. Error bars represent s.d. of mean, each measured in technical duplicates. e , Average composite rescue score of each class of proteins identified among the resistance candidates (relates to Main Fig. 2 ). Number of genes within each protein class is shown in parentheses. f , ADCY9 was identified as a resistance candidate in the primary resistance screen, but was a DNA failure in our independent prep of candidate virus. Therefore, ADCY9 was not included in the high throughput validation screens, but was included in all subsequent validation work. These data show that ADCY9 is able to confer resistance to all tested MAPK-i to a similar degree as forskolin/IBMX treatment. Error bars represent s.d. of mean, n=6 technical replicates. g , Western blot analysis of the expression of V5-epitope tagged eGFP and ADCY9 in WM266.4.

    Techniques Used: Mutagenesis, Western Blot, Infection, Expressing, High Throughput Screening Assay

    25) Product Images from "A mTurquoise-Based cAMP Sensor for Both FLIM and Ratiometric Read-Out Has Improved Dynamic Range"

    Article Title: A mTurquoise-Based cAMP Sensor for Both FLIM and Ratiometric Read-Out Has Improved Dynamic Range

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019170

    Characterization of T Epac VV as a FRET sensor for cAMP. (A) Hek293 cells expressing indicated constructs show cytosolic localization. Confocal images were taken 18 hours after transfection. (B) cAMP-induced conformational switch in T Epac VV causes drop in FRET. (C) Live-cell emission spectra of T Epac VV in rest and after stimulation with IBMX and forskolin (forsk) to saturate cytosolic cAMP levels. Spectra (average of 6 individual cells) are normalized with respect to the isosbestic point at 505 nm. (D) Performance of T Epac VV in live-cell FLIM detections. Shown is a representative time-lapse recording of τ φ and τ m displaying a large change in FRET upon addition of IBMX and forskolin. Changes are shown for four individual cells present in the same microscope image. (E) Time-lapse FLIM recordings from cells showing much increased stability of T Epac VV (dark blue) as compared to C Epac VV (violet trace). Each trace depicts 200 FLIM recordings consisting of 12 phase images for a total of 2400 images. Exposure time was 200 ms at ∼0.8 mW output power at the image plane. Traces are average of n = 6 single-cell determinations. (F) Log/linear plot of TCSPC data illustrates close-to-single-exponential decay of fluorescence in T Epac VV in rest (dark blue trace). Saturation of cAMP levels with IBMX + forskolin (bright blue trace) causes the decay time constant to increase from 2.53±0.02 to 2.97±0.03 ns in this experiment. The red trace shows distinctly bi-exponential decay of C Epac VV for comparison. Representative example of n > 12 experiments. (G) Analysis of cAMP-induced FRET changes in T Epac VV analyzed by ratiometry. Changes in emission intensity in the CFP channel (blue) and YFP channel (yellow) demonstrate the exceptional S/N and FRET span of T Epac VV . The black trace depicts the ratio CFP/YFP. Sampling interval was 0.5 s. (H) Quantification of ratiometric changes, showing the increased ratio span of our novel sensor. Data are mean ± s.e.m. from at least 12 independent determinations. (I) Typical FRET trace, obtained from a single Hek293 cell expressing T Epac VV . Following recording of a baseline, the preparation was stimulated with prostaglandin E1 (PGE1, 5 µM) and isoproterenol (10 µM) as indicated. Calibration was with IBMX + forskolin. Traces of this quality were routinely obtained. Note that all experiments were carried out at 37°C except for the frequency-domain FLIM experiments, which were done at room temperature. This temperature difference explains the approximately 50% slower risetimes of agonist-induced cAMP changes that were generally observed in frequency-domain FLIM measurements. Conversely, ratiometric detections carried out at room temperature were also slow.
    Figure Legend Snippet: Characterization of T Epac VV as a FRET sensor for cAMP. (A) Hek293 cells expressing indicated constructs show cytosolic localization. Confocal images were taken 18 hours after transfection. (B) cAMP-induced conformational switch in T Epac VV causes drop in FRET. (C) Live-cell emission spectra of T Epac VV in rest and after stimulation with IBMX and forskolin (forsk) to saturate cytosolic cAMP levels. Spectra (average of 6 individual cells) are normalized with respect to the isosbestic point at 505 nm. (D) Performance of T Epac VV in live-cell FLIM detections. Shown is a representative time-lapse recording of τ φ and τ m displaying a large change in FRET upon addition of IBMX and forskolin. Changes are shown for four individual cells present in the same microscope image. (E) Time-lapse FLIM recordings from cells showing much increased stability of T Epac VV (dark blue) as compared to C Epac VV (violet trace). Each trace depicts 200 FLIM recordings consisting of 12 phase images for a total of 2400 images. Exposure time was 200 ms at ∼0.8 mW output power at the image plane. Traces are average of n = 6 single-cell determinations. (F) Log/linear plot of TCSPC data illustrates close-to-single-exponential decay of fluorescence in T Epac VV in rest (dark blue trace). Saturation of cAMP levels with IBMX + forskolin (bright blue trace) causes the decay time constant to increase from 2.53±0.02 to 2.97±0.03 ns in this experiment. The red trace shows distinctly bi-exponential decay of C Epac VV for comparison. Representative example of n > 12 experiments. (G) Analysis of cAMP-induced FRET changes in T Epac VV analyzed by ratiometry. Changes in emission intensity in the CFP channel (blue) and YFP channel (yellow) demonstrate the exceptional S/N and FRET span of T Epac VV . The black trace depicts the ratio CFP/YFP. Sampling interval was 0.5 s. (H) Quantification of ratiometric changes, showing the increased ratio span of our novel sensor. Data are mean ± s.e.m. from at least 12 independent determinations. (I) Typical FRET trace, obtained from a single Hek293 cell expressing T Epac VV . Following recording of a baseline, the preparation was stimulated with prostaglandin E1 (PGE1, 5 µM) and isoproterenol (10 µM) as indicated. Calibration was with IBMX + forskolin. Traces of this quality were routinely obtained. Note that all experiments were carried out at 37°C except for the frequency-domain FLIM experiments, which were done at room temperature. This temperature difference explains the approximately 50% slower risetimes of agonist-induced cAMP changes that were generally observed in frequency-domain FLIM measurements. Conversely, ratiometric detections carried out at room temperature were also slow.

    Techniques Used: Expressing, Construct, Transfection, Microscopy, Mass Spectrometry, Fluorescence, Sampling

    26) Product Images from "Two microRNAs, miR-330 and miR-125b-5p, mark the juxtaglomerular cell and balance its smooth muscle phenotype"

    Article Title: Two microRNAs, miR-330 and miR-125b-5p, mark the juxtaglomerular cell and balance its smooth muscle phenotype

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00460.2011

    miRNA expression in kidney and SMCs measured by RT-PCR. Figure shows a representative agarose gel stained with ethidium bromide of RT-PCR reactions carried out using RNA templates from SMCs and kidney cortex, specific mature miRNA primers, and a universal PCR primer for miRNA (NCode kit, Invitrogen). C, control (untreated); T, treated kidney (low Na+captopril) or SMCs (forskolin+IBMX). The observed sizes of the miRNA products matched the predicted sizes, which were between 50 and 65 bp. Expression of 5S rRNA was used as an internal control.
    Figure Legend Snippet: miRNA expression in kidney and SMCs measured by RT-PCR. Figure shows a representative agarose gel stained with ethidium bromide of RT-PCR reactions carried out using RNA templates from SMCs and kidney cortex, specific mature miRNA primers, and a universal PCR primer for miRNA (NCode kit, Invitrogen). C, control (untreated); T, treated kidney (low Na+captopril) or SMCs (forskolin+IBMX). The observed sizes of the miRNA products matched the predicted sizes, which were between 50 and 65 bp. Expression of 5S rRNA was used as an internal control.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction

    Effect of miR-330 and miR-125b-5p on smooth muscle gene expression. Expression of smooth muscle genes smoothelin ( Smtn ), smooth muscle myosin heavy chain ( Myh11 ) and α-smooth muscle actin ( Acta2 ) assessed in cultured SMCs treated with forskolin+IBMX for 24 h to induce renin reexpression. Figure shows the results of quantitative ( Smtn ) and semiquantitative ( Myh11 , Acta2 ) RT-PCR experiments. All three smooth muscle genes were downregulated as the cells adopted the renin phenotype in response to forskolin+IBMX. B : expression of smoothelin in cells transfected with precursors and inhibitors of miR-330 and miR-125b-5p. SMCs were transfected with 50 nM pre-miR miRNA precursor or anti-miR miRNA inhibitor for 36 h. Transfection of SMCs with pre-miR-330 significantly reduced Smtn mRNA expression. Treatment with anti-miR-330 inhibitor (Inh330) did not alter Smtn expression compared with control levels. Pre-miR-125b-5p precursor significantly increased Smtn levels whereas anti-miR-125b-5p inhibitor reduced Smtn expression. These results suggest that miR-330 and miR-125b-5p have opposite effects in the regulation of smooth muscle expression in cells of the renin lineage. Control, nontargeting pre-miRNA; Pre-330, Pre-miR-330; Inh-330, inhibitor miR-330; Pre-125b-5p, Pre-miR-125b-5p; Inh-125b-5p, inhibitor miR-125b-5p. Values are means ± SD; n = 6. In A and B , * P
    Figure Legend Snippet: Effect of miR-330 and miR-125b-5p on smooth muscle gene expression. Expression of smooth muscle genes smoothelin ( Smtn ), smooth muscle myosin heavy chain ( Myh11 ) and α-smooth muscle actin ( Acta2 ) assessed in cultured SMCs treated with forskolin+IBMX for 24 h to induce renin reexpression. Figure shows the results of quantitative ( Smtn ) and semiquantitative ( Myh11 , Acta2 ) RT-PCR experiments. All three smooth muscle genes were downregulated as the cells adopted the renin phenotype in response to forskolin+IBMX. B : expression of smoothelin in cells transfected with precursors and inhibitors of miR-330 and miR-125b-5p. SMCs were transfected with 50 nM pre-miR miRNA precursor or anti-miR miRNA inhibitor for 36 h. Transfection of SMCs with pre-miR-330 significantly reduced Smtn mRNA expression. Treatment with anti-miR-330 inhibitor (Inh330) did not alter Smtn expression compared with control levels. Pre-miR-125b-5p precursor significantly increased Smtn levels whereas anti-miR-125b-5p inhibitor reduced Smtn expression. These results suggest that miR-330 and miR-125b-5p have opposite effects in the regulation of smooth muscle expression in cells of the renin lineage. Control, nontargeting pre-miRNA; Pre-330, Pre-miR-330; Inh-330, inhibitor miR-330; Pre-125b-5p, Pre-miR-125b-5p; Inh-125b-5p, inhibitor miR-125b-5p. Values are means ± SD; n = 6. In A and B , * P

    Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Transfection

    27) Product Images from "Myosin1D is an evolutionarily conserved regulator of animal left–right asymmetry"

    Article Title: Myosin1D is an evolutionarily conserved regulator of animal left–right asymmetry

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04284-8

    myo1D controls the morphogenesis of the zebrafish left–right Organizer. a – e Compared to WT ( n = 104), MZ myo1D mutants ( n = 118) present a reduced KV size. Treatment with IBMX and Forskolin promotes KV lumen inflation and increases organ size in WT ( n = 66) and MZ myo1D mutants ( n = 85). e Dot plots of KV equatorial surface area in individual embryos. KV size is similar in embryos with normal or defective laterality. f IBMX/Forskolin treatment restores KV size ( d , e ), but not laterality in MZ myo1D mutants. g – j Cilia number and size are reduced in MZ myo1D mutants. g , h Projection of images from confocal stacks used to quantify number and length of cilia (acetylated tubulin, magenta) in the KV (ZO-1 positive cells, green). i , j Number and average length of cilia in individual embryos are reduced in MZ myo1D ( n = 117 embryos/4939 cilia) compared to WT ( n = 141/6967). k – m Confocal imaging of Arl13b-GFP labeled cilia in living embryos was used to analyze the number ( k ), motility ( l ) and positioning ( m ) of KV cilia. k Dot plot representing motile cilia number in individual embryos. MZ myo1D ( n = 42) mutants display lower motile cilia numbers compared to WT ( n = 33). Motile cilia numbers are however similar in MZ myo1D . l myo1D loss of function does not impair ciliary motility. m WT and MZ myo1D mutant animals display a similar enrichment of cilia in the anterior KV half. a – d , g , h are dorsal views of the KV, anterior up. All data collected at the eight-somites stage. Horizontal bars in e , i , j , k represent mean values. Error bars in l , m represent SEM. Scale bars: 20 µm in a – d ; 10 µm in g – h
    Figure Legend Snippet: myo1D controls the morphogenesis of the zebrafish left–right Organizer. a – e Compared to WT ( n = 104), MZ myo1D mutants ( n = 118) present a reduced KV size. Treatment with IBMX and Forskolin promotes KV lumen inflation and increases organ size in WT ( n = 66) and MZ myo1D mutants ( n = 85). e Dot plots of KV equatorial surface area in individual embryos. KV size is similar in embryos with normal or defective laterality. f IBMX/Forskolin treatment restores KV size ( d , e ), but not laterality in MZ myo1D mutants. g – j Cilia number and size are reduced in MZ myo1D mutants. g , h Projection of images from confocal stacks used to quantify number and length of cilia (acetylated tubulin, magenta) in the KV (ZO-1 positive cells, green). i , j Number and average length of cilia in individual embryos are reduced in MZ myo1D ( n = 117 embryos/4939 cilia) compared to WT ( n = 141/6967). k – m Confocal imaging of Arl13b-GFP labeled cilia in living embryos was used to analyze the number ( k ), motility ( l ) and positioning ( m ) of KV cilia. k Dot plot representing motile cilia number in individual embryos. MZ myo1D ( n = 42) mutants display lower motile cilia numbers compared to WT ( n = 33). Motile cilia numbers are however similar in MZ myo1D . l myo1D loss of function does not impair ciliary motility. m WT and MZ myo1D mutant animals display a similar enrichment of cilia in the anterior KV half. a – d , g , h are dorsal views of the KV, anterior up. All data collected at the eight-somites stage. Horizontal bars in e , i , j , k represent mean values. Error bars in l , m represent SEM. Scale bars: 20 µm in a – d ; 10 µm in g – h

    Techniques Used: Imaging, Labeling, Mutagenesis

    28) Product Images from "Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes"

    Article Title: Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes

    Journal: eLife

    doi: 10.7554/eLife.01133

    Sgol2-PP2A interaction is required to recruit PP2A to kinetochores and protect centromeric cohesion at the first meiotic division. ( A ) GV oocytes were harvested in the presence of IBMX. Microinjections were performed at GV stage in M2 medium supplemented with IBMX. Oocytes were then cultured in IBMX-free medium for about 6 hr, corresponding to metaphase I stage. Chromosome spreads were prepared and stained with DAPI (blue), Sgol2 (red), and PP2A-C (green). Box and whisker plot shows fluorescence intensity ratios of PP2A and Sgol2 at kinetochores. Upper and lower bars indicate 95th and 5th percentiles, respectively. Numbers of oocytes examined are indicated (n). ( B ) Oocytes harvested at GV stage were matured in M16 medium for up to 24 hr. Chromosome spreads prepared from oocytes that have extruded the first polar body were stained with DAPI (blue) to visualize DNA and CREST (green) to mark centromeres. Frequencies of dyads and single chromatids were quantified. DOI: http://dx.doi.org/10.7554/eLife.01133.004
    Figure Legend Snippet: Sgol2-PP2A interaction is required to recruit PP2A to kinetochores and protect centromeric cohesion at the first meiotic division. ( A ) GV oocytes were harvested in the presence of IBMX. Microinjections were performed at GV stage in M2 medium supplemented with IBMX. Oocytes were then cultured in IBMX-free medium for about 6 hr, corresponding to metaphase I stage. Chromosome spreads were prepared and stained with DAPI (blue), Sgol2 (red), and PP2A-C (green). Box and whisker plot shows fluorescence intensity ratios of PP2A and Sgol2 at kinetochores. Upper and lower bars indicate 95th and 5th percentiles, respectively. Numbers of oocytes examined are indicated (n). ( B ) Oocytes harvested at GV stage were matured in M16 medium for up to 24 hr. Chromosome spreads prepared from oocytes that have extruded the first polar body were stained with DAPI (blue) to visualize DNA and CREST (green) to mark centromeres. Frequencies of dyads and single chromatids were quantified. DOI: http://dx.doi.org/10.7554/eLife.01133.004

    Techniques Used: Cell Culture, Staining, Whisker Assay, Fluorescence

    GV oocytes were harvested in M2 medium supplemented with IBMX. Oocytes were then cultured in IBMX-free M16 medium for about 6–7 hr, corresponding to metaphase I-stage. To inhibit Aurora B/C kinase activity oocytes were cultured in M16 medium supplemented with AZD1152 (100 nM). Representative chromosomes spreads from indicated groups of oocytes at metaphase I-stage showing MCAK localization are displayed. DOI: http://dx.doi.org/10.7554/eLife.01133.011
    Figure Legend Snippet: GV oocytes were harvested in M2 medium supplemented with IBMX. Oocytes were then cultured in IBMX-free M16 medium for about 6–7 hr, corresponding to metaphase I-stage. To inhibit Aurora B/C kinase activity oocytes were cultured in M16 medium supplemented with AZD1152 (100 nM). Representative chromosomes spreads from indicated groups of oocytes at metaphase I-stage showing MCAK localization are displayed. DOI: http://dx.doi.org/10.7554/eLife.01133.011

    Techniques Used: Cell Culture, Activity Assay

    Sgol2-PP2A, but not Sgol2-Mad2, interaction is required to protect centromeric cohesion at the first meiotic division. ( A ) Oocytes harvested at GV stage from wild type (control) mice were microinjected with wild type or 3A mutant Sgol2 mRNA in M2 medium supplemented with IBMX. After releasing the oocytes in M16 medium without IBMX, chromosome spreads were prepared from metaphase II-arrested oocytes and stained with DAPI (blue) and CREST (green). ( B ) Frequencies of dyads and single chromatids observed on metaphase II-stage chromosome spreads. Numbers of oocytes examined are indicated (n). ( C ) Metaphase I localization of Sgol2 (red) and PP2A-C (green) on chromosomes from sgol2 Δ/Δ oocytes microinjected with H150E Sgol2 mRNA. ( D ) DAPI (blue) and CREST (green) stained chromosome spreads prepared from metaphase II-stage oocytes. DOI: http://dx.doi.org/10.7554/eLife.01133.005
    Figure Legend Snippet: Sgol2-PP2A, but not Sgol2-Mad2, interaction is required to protect centromeric cohesion at the first meiotic division. ( A ) Oocytes harvested at GV stage from wild type (control) mice were microinjected with wild type or 3A mutant Sgol2 mRNA in M2 medium supplemented with IBMX. After releasing the oocytes in M16 medium without IBMX, chromosome spreads were prepared from metaphase II-arrested oocytes and stained with DAPI (blue) and CREST (green). ( B ) Frequencies of dyads and single chromatids observed on metaphase II-stage chromosome spreads. Numbers of oocytes examined are indicated (n). ( C ) Metaphase I localization of Sgol2 (red) and PP2A-C (green) on chromosomes from sgol2 Δ/Δ oocytes microinjected with H150E Sgol2 mRNA. ( D ) DAPI (blue) and CREST (green) stained chromosome spreads prepared from metaphase II-stage oocytes. DOI: http://dx.doi.org/10.7554/eLife.01133.005

    Techniques Used: Mouse Assay, Mutagenesis, Staining

    29) Product Images from "miR-425-5p Inhibits Differentiation and Proliferation in Porcine Intramuscular Preadipocytes"

    Article Title: miR-425-5p Inhibits Differentiation and Proliferation in Porcine Intramuscular Preadipocytes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18102101

    Expression profile of miR-425-5p in different porcine tissues and during porcine intramuscular preadipocyte differentiation. ( A ) Mature miR-425-5p sequence was conserved among species; ( B ) expression of miR-425-5p was analyzed by real-time PCR in seven different tissues of 180-day old Guanzhong Black pigs; ( C ) photomicrographs showing cell morphology change during porcine intramuscular preadipocyte differentiation (( a ) porcine intramuscular preadipocyte before 3-isobutyl-1-methylxanthine (IBMX)–Dexamethasone (DEX)–insulin (DMI) induction; ( b ) porcine intramuscular preadipocyte 2 days after DMI induction; ( c ) porcine intramuscular preadipocyte 8 days after DMI induction; ( d ) porcine intramuscular preadipocyte 8 days after DMI induction staining with Oil Red O); ( D ) expression of miR-425-5p during porcine intramuscular preadipocyte differentiation and the expression at the day 0 as a control. U6 small nuclear RNA was used as a reference gene. Results were presented as means ± SEM, n = 3; ** p
    Figure Legend Snippet: Expression profile of miR-425-5p in different porcine tissues and during porcine intramuscular preadipocyte differentiation. ( A ) Mature miR-425-5p sequence was conserved among species; ( B ) expression of miR-425-5p was analyzed by real-time PCR in seven different tissues of 180-day old Guanzhong Black pigs; ( C ) photomicrographs showing cell morphology change during porcine intramuscular preadipocyte differentiation (( a ) porcine intramuscular preadipocyte before 3-isobutyl-1-methylxanthine (IBMX)–Dexamethasone (DEX)–insulin (DMI) induction; ( b ) porcine intramuscular preadipocyte 2 days after DMI induction; ( c ) porcine intramuscular preadipocyte 8 days after DMI induction; ( d ) porcine intramuscular preadipocyte 8 days after DMI induction staining with Oil Red O); ( D ) expression of miR-425-5p during porcine intramuscular preadipocyte differentiation and the expression at the day 0 as a control. U6 small nuclear RNA was used as a reference gene. Results were presented as means ± SEM, n = 3; ** p

    Techniques Used: Expressing, Sequencing, Real-time Polymerase Chain Reaction, Staining

    30) Product Images from "Suppression of PU.1-linked TLR4 expression by cilostazol with decrease of cytokine production in macrophages from patients with rheumatoid arthritis"

    Article Title: Suppression of PU.1-linked TLR4 expression by cilostazol with decrease of cytokine production in macrophages from patients with rheumatoid arthritis

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.12021

    Inhibitory effects of cilostazol and forskolin/IBMX on LPS-induced TLR4 mRNA and protein expression in RA macrophages. (A) Intracellular cAMP level in RA macrophages after treatment with cilostazol (1–100 μM) or forskolin (10 μM)
    Figure Legend Snippet: Inhibitory effects of cilostazol and forskolin/IBMX on LPS-induced TLR4 mRNA and protein expression in RA macrophages. (A) Intracellular cAMP level in RA macrophages after treatment with cilostazol (1–100 μM) or forskolin (10 μM)

    Techniques Used: Expressing

    Effects of cilostazol (CSZ) (10 μM) and forskolin (10 μM) +IBMX (100 μM) on TNF-α (A), IL-1β (B) and IL-10 (C) levels that were released into the incubation media of RA macrophages following LPS treatment. (D) Inhibition
    Figure Legend Snippet: Effects of cilostazol (CSZ) (10 μM) and forskolin (10 μM) +IBMX (100 μM) on TNF-α (A), IL-1β (B) and IL-10 (C) levels that were released into the incubation media of RA macrophages following LPS treatment. (D) Inhibition

    Techniques Used: Incubation, Inhibition

    Effect of cilostazol and forskolin + IBMX on LPS-stimulated IκBα degradation in the cytoplasm and NF-κB p65 expression in the nucleus following LPS stimulation. (A) Treatment with cilostazol and forskolin + IBMX for 4 h increased
    Figure Legend Snippet: Effect of cilostazol and forskolin + IBMX on LPS-stimulated IκBα degradation in the cytoplasm and NF-κB p65 expression in the nucleus following LPS stimulation. (A) Treatment with cilostazol and forskolin + IBMX for 4 h increased

    Techniques Used: Expressing

    31) Product Images from "Inhibitory effect of caffeine on pacemaker activity in the oviduct is mediated by cAMP-regulated conductances"

    Article Title: Inhibitory effect of caffeine on pacemaker activity in the oviduct is mediated by cAMP-regulated conductances

    Journal: British Journal of Pharmacology

    doi: 10.1111/j.1476-5381.2011.01266.x

    Inhibition of myosalpinx slow waves by IBMX was antagonized by glibenclamide. (A) An intracellular microelectrode recording showing the effects of the PDE inhibitor IBMX (10 µM) on slow wave activity. Application of IBMX caused membrane hyperpolarization
    Figure Legend Snippet: Inhibition of myosalpinx slow waves by IBMX was antagonized by glibenclamide. (A) An intracellular microelectrode recording showing the effects of the PDE inhibitor IBMX (10 µM) on slow wave activity. Application of IBMX caused membrane hyperpolarization

    Techniques Used: Inhibition, Activity Assay

    32) Product Images from "APC/CCdh1 Enables Removal of Shugoshin-2 from the Arms of Bivalent Chromosomes by Moderating Cyclin-Dependent Kinase Activity"

    Article Title: APC/CCdh1 Enables Removal of Shugoshin-2 from the Arms of Bivalent Chromosomes by Moderating Cyclin-Dependent Kinase Activity

    Journal: Current Biology

    doi: 10.1016/j.cub.2017.04.023

    Following GVBD, Sgol2 Localizes on the Chromosome Arms and Kinetochores, and It Gradually Concentrates on Kinetochores during Late Metaphase (A) GV-stage oocytes harvested from Sgol2 -deleted females were microinjected in M2 medium supplemented with IBMX with mRNA encoding GFP-Sgol2 and H2B-mCherry. After 1 hr of incubation, oocytes were released, and time-lapse confocal microscope images were captured for 12–14 hr following GVBD. Representative Z-projected images are displayed. (B) GV-stage oocytes harvested from wild-type control females were cultured for 2, 4, and 6 hr following GVBD. Chromosome spreads prepared at the indicated times following GVBD were stained for DNA (blue), Sgol2 (red), and CREST (green). See also Figure S2 .
    Figure Legend Snippet: Following GVBD, Sgol2 Localizes on the Chromosome Arms and Kinetochores, and It Gradually Concentrates on Kinetochores during Late Metaphase (A) GV-stage oocytes harvested from Sgol2 -deleted females were microinjected in M2 medium supplemented with IBMX with mRNA encoding GFP-Sgol2 and H2B-mCherry. After 1 hr of incubation, oocytes were released, and time-lapse confocal microscope images were captured for 12–14 hr following GVBD. Representative Z-projected images are displayed. (B) GV-stage oocytes harvested from wild-type control females were cultured for 2, 4, and 6 hr following GVBD. Chromosome spreads prepared at the indicated times following GVBD were stained for DNA (blue), Sgol2 (red), and CREST (green). See also Figure S2 .

    Techniques Used: Incubation, Microscopy, Cell Culture, Staining

    33) Product Images from "Effect of xanthohumol on melanogenesis in B16 melanoma cells"

    Article Title: Effect of xanthohumol on melanogenesis in B16 melanoma cells

    Journal:

    doi: 10.3858/emm.2008.40.3.313

    Effect of XH on α-MSH-, IBMX- and forskolin-induced melanogenesis. (A) Cells (5 × 10 6 ) were incubated with 5 µM XH in the presence of IBMX (0.1 mM), α-MSH (5 µM), or forskolin (5 µM) for 2 days. Melanin
    Figure Legend Snippet: Effect of XH on α-MSH-, IBMX- and forskolin-induced melanogenesis. (A) Cells (5 × 10 6 ) were incubated with 5 µM XH in the presence of IBMX (0.1 mM), α-MSH (5 µM), or forskolin (5 µM) for 2 days. Melanin

    Techniques Used: Incubation

    34) Product Images from "Adaptive downregulation of Cl-/HCO3- exchange activity in rat hepatocytes under experimental obstructive cholestasis"

    Article Title: Adaptive downregulation of Cl-/HCO3- exchange activity in rat hepatocytes under experimental obstructive cholestasis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0212215

    Changes in hepatocyte anion exchange activity induced by BDL. (A) Representative traces of changes in intracellular pH (pHi) of perfused rat hepatocytes showing the recovering acidification that follows the alkalinization elicited by propionate removal, and results from AE2-mediated HCO 3 - secretion (demarcated zone in the curves). Inset graph shows the pH i changes with time, indicative of AE activity, during the phase of recovering acidification, expressed as the percent of the initial, maximum pH i in BDL and SHAM hepatocytes, under the presence or absence of a stimulatory mixture (SM: dibutyryl cyclic AMP, IBMX and forskolin) in the perfusion buffers. (B) Anion exchange (AE) activity, expressed as J OH -, in primary cultured hepatocytes from BDL and SHAM rats, in the presence or absence of SM in the perfusion buffers. Results are mean ± SEM, n = 20 to 60 cells per preparation, from 3 independent cellular preparations per experimental group.
    Figure Legend Snippet: Changes in hepatocyte anion exchange activity induced by BDL. (A) Representative traces of changes in intracellular pH (pHi) of perfused rat hepatocytes showing the recovering acidification that follows the alkalinization elicited by propionate removal, and results from AE2-mediated HCO 3 - secretion (demarcated zone in the curves). Inset graph shows the pH i changes with time, indicative of AE activity, during the phase of recovering acidification, expressed as the percent of the initial, maximum pH i in BDL and SHAM hepatocytes, under the presence or absence of a stimulatory mixture (SM: dibutyryl cyclic AMP, IBMX and forskolin) in the perfusion buffers. (B) Anion exchange (AE) activity, expressed as J OH -, in primary cultured hepatocytes from BDL and SHAM rats, in the presence or absence of SM in the perfusion buffers. Results are mean ± SEM, n = 20 to 60 cells per preparation, from 3 independent cellular preparations per experimental group.

    Techniques Used: Activity Assay, Cell Culture

    35) Product Images from "Vesicle-associated Membrane Protein 3 (VAMP3) Mediates Constitutive Trafficking of the Renal Co-transporter NKCC2 in Thick Ascending Limbs"

    Article Title: Vesicle-associated Membrane Protein 3 (VAMP3) Mediates Constitutive Trafficking of the Renal Co-transporter NKCC2 in Thick Ascending Limbs

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.735167

    Silencing VAMP3 decreases constitutive steady-state surface NKCC2 expression in rat TALs. A , representative surface biotinylation experiment showing steady-state surface and intracellular NKCC2 in TALs from rats transduced in vivo with VAMP3-shRNA or scrambled-shRNA. TALs were treated with vehicle or forskolin + IBMX to stimulate cAMP. Intracellular protein GAPDH was not detected at the surface. B , quantification of steady-state surface NKCC2 after silencing VAMP3 as measured by surface biotinylation in TALs. In VAMP3-shRNA transduced TALs, the constitutive (vehicle) surface to intracellular NKCC2 ratio was decreased by 27% ( gray bars ), and steady-state surface NKCC2 was reduced by 43% ± 7% ( black bars ). However, cAMP increased steady-state surface NKCC2 expression equally in scrambled-shRNA and VAMP3-shRNA-transduced TALs. The values represent the averages in each experimental group. Error bars represent ± S.E. *, p
    Figure Legend Snippet: Silencing VAMP3 decreases constitutive steady-state surface NKCC2 expression in rat TALs. A , representative surface biotinylation experiment showing steady-state surface and intracellular NKCC2 in TALs from rats transduced in vivo with VAMP3-shRNA or scrambled-shRNA. TALs were treated with vehicle or forskolin + IBMX to stimulate cAMP. Intracellular protein GAPDH was not detected at the surface. B , quantification of steady-state surface NKCC2 after silencing VAMP3 as measured by surface biotinylation in TALs. In VAMP3-shRNA transduced TALs, the constitutive (vehicle) surface to intracellular NKCC2 ratio was decreased by 27% ( gray bars ), and steady-state surface NKCC2 was reduced by 43% ± 7% ( black bars ). However, cAMP increased steady-state surface NKCC2 expression equally in scrambled-shRNA and VAMP3-shRNA-transduced TALs. The values represent the averages in each experimental group. Error bars represent ± S.E. *, p

    Techniques Used: Expressing, In Vivo, shRNA

    Silencing VAMP3 blocks constitutive NKCC2 exocytic delivery in rat TALs. A , representative Western blot showing masking of surface biotinylation sites by NHS-acetate at 4 °C and reappearance of surface NKCC2 signal after exocytic delivery at 37 °C in TALs transduced with scrambled or VAMP3-shRNA. TALs were treated with vehicle or forskolin + IBMX to stimulate cAMP. The vertical division line separates non-consecutive lanes in the same gel and film. B , quantification of NKCC2 exocytic delivery at 30 min in rat TALs measured as biotinylated NKCC2 at the surface after masking with NHS-acetate. In rats transduced in vivo with VAMP3-shRNA, constitutive NKCC2 exocytic delivery was decreased by 86% after silencing VAMP3. However, cAMP was still able to stimulate NKCC2 exocytic delivery after silencing VAMP3. For every experiment (scrambled-shRNA and VAMP3-shRNA), the difference between the non-NHS-acetate masked fraction and the NHS-acetate masked fraction at time 0 was used as reference to calculate the NHS-acetate masked fraction. The values represent the mean percentages of NHS-acetate masked fraction. Error bars represent ± S.E. *, p
    Figure Legend Snippet: Silencing VAMP3 blocks constitutive NKCC2 exocytic delivery in rat TALs. A , representative Western blot showing masking of surface biotinylation sites by NHS-acetate at 4 °C and reappearance of surface NKCC2 signal after exocytic delivery at 37 °C in TALs transduced with scrambled or VAMP3-shRNA. TALs were treated with vehicle or forskolin + IBMX to stimulate cAMP. The vertical division line separates non-consecutive lanes in the same gel and film. B , quantification of NKCC2 exocytic delivery at 30 min in rat TALs measured as biotinylated NKCC2 at the surface after masking with NHS-acetate. In rats transduced in vivo with VAMP3-shRNA, constitutive NKCC2 exocytic delivery was decreased by 86% after silencing VAMP3. However, cAMP was still able to stimulate NKCC2 exocytic delivery after silencing VAMP3. For every experiment (scrambled-shRNA and VAMP3-shRNA), the difference between the non-NHS-acetate masked fraction and the NHS-acetate masked fraction at time 0 was used as reference to calculate the NHS-acetate masked fraction. The values represent the mean percentages of NHS-acetate masked fraction. Error bars represent ± S.E. *, p

    Techniques Used: Western Blot, Transduction, shRNA, In Vivo

    36) Product Images from "Activating PRKACB somatic mutation in cortisol-producing adenomas"

    Article Title: Activating PRKACB somatic mutation in cortisol-producing adenomas

    Journal: JCI Insight

    doi: 10.1172/jci.insight.98296

    Mutant type I PKA holoenzyme stability is decreased in HEK293 cells. Bioluminescence resonance energy transfer (BRET) experiments were used to test PKA type I ( A ) and type II ( B ) holoenzyme formation and dissociation. HEK293 cells were cotransfected with RLuc-tagged regulatory subunits (RIα or RIβ in panel A and RIIα or RIIβ in panel B ) as a donor (emission 410 nm) and GFP-tagged catalytic subunits (wild-type [WT] or mutant [S54L] Cβ isoform 1) as an acceptor (emission 515 nm). Cells were treated either with buffer (basal condition, black boxes) or with 50 μM adenylyl cyclase activator forskolin (Fsk) and 100 μM phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to induce holoenzyme dissociation (stimulated condition, white boxes). Transfection of the empty RLuc8 vector was used as control (Ctrl). ( A ) The BRET ratio is significantly lower in basal and stimulated conditions for type I holoenzymes with the S54L mutant compared with the WT, indicating a decrease in mutant holoenzyme formation and stability. ( B ) No significant differences in BRET ratio were observed under basal conditions for the type II holoenzymes, indicating that the S54L mutation does not disturb the holoenzyme formed with type II regulatory subunits. Data from 3–6 replicates from 3 independent experiments are represented as dot plots and analyzed by unpaired t test with Sidak’s post hoc test for multiple comparisons. * P
    Figure Legend Snippet: Mutant type I PKA holoenzyme stability is decreased in HEK293 cells. Bioluminescence resonance energy transfer (BRET) experiments were used to test PKA type I ( A ) and type II ( B ) holoenzyme formation and dissociation. HEK293 cells were cotransfected with RLuc-tagged regulatory subunits (RIα or RIβ in panel A and RIIα or RIIβ in panel B ) as a donor (emission 410 nm) and GFP-tagged catalytic subunits (wild-type [WT] or mutant [S54L] Cβ isoform 1) as an acceptor (emission 515 nm). Cells were treated either with buffer (basal condition, black boxes) or with 50 μM adenylyl cyclase activator forskolin (Fsk) and 100 μM phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to induce holoenzyme dissociation (stimulated condition, white boxes). Transfection of the empty RLuc8 vector was used as control (Ctrl). ( A ) The BRET ratio is significantly lower in basal and stimulated conditions for type I holoenzymes with the S54L mutant compared with the WT, indicating a decrease in mutant holoenzyme formation and stability. ( B ) No significant differences in BRET ratio were observed under basal conditions for the type II holoenzymes, indicating that the S54L mutation does not disturb the holoenzyme formed with type II regulatory subunits. Data from 3–6 replicates from 3 independent experiments are represented as dot plots and analyzed by unpaired t test with Sidak’s post hoc test for multiple comparisons. * P

    Techniques Used: Mutagenesis, Bioluminescence Resonance Energy Transfer, Transfection, Plasmid Preparation

    37) Product Images from "Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3"

    Article Title: Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3

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

    doi: 10.1073/pnas.1312701111

    S910 und S935 are both PKA phosphorylation sites in LRRK2. ( A ) StrepII/Flag-tagged LRRK2 WT purified from an insect cell expression system was phosphorylated by PKA in vitro. Products were separated on 4–12% gradient gels and analyzed by Western blot using phosphospecific antibodies against pS910 and pS935. ( B ) Forty-eight hours after transfection with StrepII/Flag-tagged LRRK2 WT, COS7 cells were serum starved for 4 h and subsequently incubated with 50 µM FSK and 100 µM IBMX for 30 min at 37 °C. LRRK2 subsequently was enriched with Strep-Tactin Superflow, separated by SDS/PAGE, and immunoblotted with anti-p910 or anti-p935 antibodies. Amido Black stain confirmed equal protein loading.
    Figure Legend Snippet: S910 und S935 are both PKA phosphorylation sites in LRRK2. ( A ) StrepII/Flag-tagged LRRK2 WT purified from an insect cell expression system was phosphorylated by PKA in vitro. Products were separated on 4–12% gradient gels and analyzed by Western blot using phosphospecific antibodies against pS910 and pS935. ( B ) Forty-eight hours after transfection with StrepII/Flag-tagged LRRK2 WT, COS7 cells were serum starved for 4 h and subsequently incubated with 50 µM FSK and 100 µM IBMX for 30 min at 37 °C. LRRK2 subsequently was enriched with Strep-Tactin Superflow, separated by SDS/PAGE, and immunoblotted with anti-p910 or anti-p935 antibodies. Amido Black stain confirmed equal protein loading.

    Techniques Used: Purification, Expressing, In Vitro, Western Blot, Transfection, Incubation, SDS Page, Staining

    38) Product Images from "Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor"

    Article Title: Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor

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

    doi: 10.1073/pnas.1902825116

    OPN3 modulates MC1R signaling. ( A ) OPN3 inhibits the MC1R-evoked cAMP response via a PTX-sensitive mechanism in MNT-1 cells. ( A , i ) OPN3 inhibits the α-MSH–induced cAMP responses of MC1R. MNT-1 cells expressing the FRET-based cAMP indicator Epac H187 and OPN3-cMCh or MCh alone (CTRL) were stimulated with α-MSH (1 μM). The cAMP response of individual cells was monitored as the ratio of CFP and YFP fluorescence intensities and represented as a function of time. FSK and IBMX were added to elicit a maximal cAMP response, used for normalization. α-MSH elicits a significant cAMP response in cells expressing MCh (CTRL, red trace), but not in cells expressing OPN3-cMCh (dark blue trace) ( n = 5–10 cells, mean ± SEM). A.U., arbitrary units. ( A , ii ) OPN3 specifically attenuates the MC1R-mediated cAMP response. Prostaglandin (PGD; 5 μM)-mediated activation of the endogenous prostaglandin E2 receptor leads to an increase in cellular cAMP (red trace) that is not attenuated in the presence of OPN3-cMCh (dark blue trace) ( n = 5–10 cells, mean ± SEM). ( A , iii ) OPN3-mediated attenuation of MC1R signaling is PTX-sensitive. MNT-1 cells treated with PTX (200 ng/mL, 4 h), which specifically inhibits the Gαi subunit of G proteins, and stimulated with α-MSH exhibited a similar cAMP response both in cells expressing MCh (red trace) and OPN3-cMCh (dark blue trace) ( n = 5–10 cells, mean ± SEM). ( A , iv ) OPN3 inhibits MC1R signaling specifically and in a Gαi-dependent manner. Average normalized amplitudes of α-MSH– or PGD-induced cAMP responses in the presence or absence of PTX show that OPN3 expression significantly reduces the amplitude of cAMP responses to α-MSH, but not to PGD, and this effect is prevented by blocking Gαi activation with PTX ( n = 3 independent experiments per condition, mean ± SEM; * P
    Figure Legend Snippet: OPN3 modulates MC1R signaling. ( A ) OPN3 inhibits the MC1R-evoked cAMP response via a PTX-sensitive mechanism in MNT-1 cells. ( A , i ) OPN3 inhibits the α-MSH–induced cAMP responses of MC1R. MNT-1 cells expressing the FRET-based cAMP indicator Epac H187 and OPN3-cMCh or MCh alone (CTRL) were stimulated with α-MSH (1 μM). The cAMP response of individual cells was monitored as the ratio of CFP and YFP fluorescence intensities and represented as a function of time. FSK and IBMX were added to elicit a maximal cAMP response, used for normalization. α-MSH elicits a significant cAMP response in cells expressing MCh (CTRL, red trace), but not in cells expressing OPN3-cMCh (dark blue trace) ( n = 5–10 cells, mean ± SEM). A.U., arbitrary units. ( A , ii ) OPN3 specifically attenuates the MC1R-mediated cAMP response. Prostaglandin (PGD; 5 μM)-mediated activation of the endogenous prostaglandin E2 receptor leads to an increase in cellular cAMP (red trace) that is not attenuated in the presence of OPN3-cMCh (dark blue trace) ( n = 5–10 cells, mean ± SEM). ( A , iii ) OPN3-mediated attenuation of MC1R signaling is PTX-sensitive. MNT-1 cells treated with PTX (200 ng/mL, 4 h), which specifically inhibits the Gαi subunit of G proteins, and stimulated with α-MSH exhibited a similar cAMP response both in cells expressing MCh (red trace) and OPN3-cMCh (dark blue trace) ( n = 5–10 cells, mean ± SEM). ( A , iv ) OPN3 inhibits MC1R signaling specifically and in a Gαi-dependent manner. Average normalized amplitudes of α-MSH– or PGD-induced cAMP responses in the presence or absence of PTX show that OPN3 expression significantly reduces the amplitude of cAMP responses to α-MSH, but not to PGD, and this effect is prevented by blocking Gαi activation with PTX ( n = 3 independent experiments per condition, mean ± SEM; * P

    Techniques Used: Expressing, Fluorescence, Activation Assay, Blocking Assay

    39) Product Images from "Redistribution of Mitochondria Leads to Bursts of ATP Production During Spontaneous Mouse Oocyte Maturation"

    Article Title: Redistribution of Mitochondria Leads to Bursts of ATP Production During Spontaneous Mouse Oocyte Maturation

    Journal: Journal of Cellular Physiology

    doi: 10.1002/jcp.22171

    The effect of cytoskeleton or enucleation on the ATP dynamic during maturation. All graphs of ATP changes were generated from regression residual data as described in Figure 1 . Both enucleation (n = 12) and nocodazole (10 µM, n = 31) completely removed the third ATP jump that occurs around 8–10 h after release from IBMX inhibition, but did not affect the first two jumps of ATP. However, ATP jumps in most of oocytes (75 ± 4.8%, 27/36) treated with cytochalasin B (10 µg/ml) were greatly reduced or abolished. In contrast to nocodazole or enucleation treatment, a smaller third ATP jump was seen in some cytochalasin treated oocytes (14/36). The right part of photos showed that both enucleation and cytochalsin B treatment did not affect the translocation to the nuclear area, but nocodazole removed the mitochondrial peri-nuclear/spindle ring. [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com .]
    Figure Legend Snippet: The effect of cytoskeleton or enucleation on the ATP dynamic during maturation. All graphs of ATP changes were generated from regression residual data as described in Figure 1 . Both enucleation (n = 12) and nocodazole (10 µM, n = 31) completely removed the third ATP jump that occurs around 8–10 h after release from IBMX inhibition, but did not affect the first two jumps of ATP. However, ATP jumps in most of oocytes (75 ± 4.8%, 27/36) treated with cytochalasin B (10 µg/ml) were greatly reduced or abolished. In contrast to nocodazole or enucleation treatment, a smaller third ATP jump was seen in some cytochalasin treated oocytes (14/36). The right part of photos showed that both enucleation and cytochalsin B treatment did not affect the translocation to the nuclear area, but nocodazole removed the mitochondrial peri-nuclear/spindle ring. [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com .]

    Techniques Used: Generated, Inhibition, Translocation Assay

    40) Product Images from "PKA Phosphorylation of NDE1 Is DISC1/PDE4 Dependent and Modulates Its Interaction with LIS1 and NDEL1"

    Article Title: PKA Phosphorylation of NDE1 Is DISC1/PDE4 Dependent and Modulates Its Interaction with LIS1 and NDEL1

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.5410-10.2011

    Effect of PKA phosphorylation of NDE1 on NDE1/LIS1 and NDE1/NDEL1 protein interactions. A , COS7 cells were transfected with V5-tagged NDE1, either wild type or mutant, and treated with IBMX plus forskolin. Top, Coimmunoprecipitation of endogenous LIS1.
    Figure Legend Snippet: Effect of PKA phosphorylation of NDE1 on NDE1/LIS1 and NDE1/NDEL1 protein interactions. A , COS7 cells were transfected with V5-tagged NDE1, either wild type or mutant, and treated with IBMX plus forskolin. Top, Coimmunoprecipitation of endogenous LIS1.

    Techniques Used: Transfection, Mutagenesis

    Related Articles

    Staining:

    Article Title: Interaction between von Hippel-Lindau Protein and Fatty Acid Synthase Modulates Hypoxia Target Gene Expression
    Article Snippet: .. 3T3-L1 cells (ATCC, CL-173) were cultured in DMEM and differentiated with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (Sigma-Aldrich, I-6634, D-4902, and I-5879 respectively), and stained with Oil Red O (Sigma-Aldrich, O-0625) as previously reported . .. MG132 (C-2211) and 25-hydroxycholesterol (H1015) were purchased from Sigma-Aldrich.

    Incubation:

    Article Title: Prolyl Isomerase Pin1 Regulates Mouse Embryonic Fibroblast Differentiation into Adipose Cells
    Article Snippet: .. Differentiation of Cells NIH3T3-L1 (control and Pin1-knockdown) and MEFs (wild-type and Pin1−/− ) were incubated in DMEM medium, containing 0.5 mM 3-isobutyl- 1-methylxanthine (Nakalai), 1 µM dexamethasone (Sigma) and 1.7 µM insulin (Wako) for 48 hours. .. Cells were cultured in insulin-containing medium for 48 hours.

    Recombinant:

    Article Title: Isolation of alveolar epithelial type II progenitor cells from adult human lungs
    Article Snippet: .. After 4 h, the media was changed to DMEM containing 5% FBS with 10 ng/ml human recombinant keratinocyte growth factor (KGF; PeproTech, Rocky Hill, NJ, USA), 0.1 mM 8-bromo cAMP (cAMP; Sigma-Aldrich) and 0.1 mM IBMX (Sigma-Aldrich). .. For hematoxylin–eosin staining, whole cell-embedded gels were fixed with 10% formalin and embedded with paraffin.

    other:

    Article Title: Intracellular potentiation between two second messenger systems may contribute to cholera toxin induced intestinal secretion in humans
    Article Snippet: ACh chloride, barium chloride, cAMP, dimethyl sulphoxide, forskolin, histamine dihydrochloride, hyoscine hydrobromide, IBMX, charybdotoxin, apamin, and vasoactive intestinal peptide were all obtained from Sigma (UK).

    Cell Culture:

    Article Title: Interaction between von Hippel-Lindau Protein and Fatty Acid Synthase Modulates Hypoxia Target Gene Expression
    Article Snippet: .. 3T3-L1 cells (ATCC, CL-173) were cultured in DMEM and differentiated with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (Sigma-Aldrich, I-6634, D-4902, and I-5879 respectively), and stained with Oil Red O (Sigma-Aldrich, O-0625) as previously reported . .. MG132 (C-2211) and 25-hydroxycholesterol (H1015) were purchased from Sigma-Aldrich.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore ibmx
    The <t>cAMP</t> response to PGE 1 is higher under the plasma membrane than in the bulk cytosol. (A) Wide-field images of a representative HEK293 cell cotransfected with R-CFP (top left) and C-YFP (not depicted) and with mp R-CFP (bottom left) and C-YFP (not depicted). For the same cell, the pseudocolor images of the 480/545-nm emission ratio before (time = 0 s) and after the addition of 10 μM PGE 1 (time = 200 s) and 100 μM <t>IBMX</t> (time = 800 s) are shown on the right. Bars, 10 μm. (B) Kinetics of cAMP changes recorded in the cells shown in A. Open circles represent kinetics recorded in the bulk cytosol with PKA-GFP, and closed circles represent kinetics recorded at the plasma membrane with mp PKA-GFP. (C) Summary of all the experiments performed in the same conditions as in A and B. (D) Time to reach half-maximal response (t/2) to 10 μM PGE 1 . (E) The summary of experiments performed by applying 1 μM PGE 1 . Error bars indicate SEM. *, 0.01
    Ibmx, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ibmx/product/Millipore
    Average 99 stars, based on 64 article reviews
    Price from $9.99 to $1999.99
    ibmx - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    The cAMP response to PGE 1 is higher under the plasma membrane than in the bulk cytosol. (A) Wide-field images of a representative HEK293 cell cotransfected with R-CFP (top left) and C-YFP (not depicted) and with mp R-CFP (bottom left) and C-YFP (not depicted). For the same cell, the pseudocolor images of the 480/545-nm emission ratio before (time = 0 s) and after the addition of 10 μM PGE 1 (time = 200 s) and 100 μM IBMX (time = 800 s) are shown on the right. Bars, 10 μm. (B) Kinetics of cAMP changes recorded in the cells shown in A. Open circles represent kinetics recorded in the bulk cytosol with PKA-GFP, and closed circles represent kinetics recorded at the plasma membrane with mp PKA-GFP. (C) Summary of all the experiments performed in the same conditions as in A and B. (D) Time to reach half-maximal response (t/2) to 10 μM PGE 1 . (E) The summary of experiments performed by applying 1 μM PGE 1 . Error bars indicate SEM. *, 0.01

    Journal: The Journal of Cell Biology

    Article Title: PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases

    doi: 10.1083/jcb.200605050

    Figure Lengend Snippet: The cAMP response to PGE 1 is higher under the plasma membrane than in the bulk cytosol. (A) Wide-field images of a representative HEK293 cell cotransfected with R-CFP (top left) and C-YFP (not depicted) and with mp R-CFP (bottom left) and C-YFP (not depicted). For the same cell, the pseudocolor images of the 480/545-nm emission ratio before (time = 0 s) and after the addition of 10 μM PGE 1 (time = 200 s) and 100 μM IBMX (time = 800 s) are shown on the right. Bars, 10 μm. (B) Kinetics of cAMP changes recorded in the cells shown in A. Open circles represent kinetics recorded in the bulk cytosol with PKA-GFP, and closed circles represent kinetics recorded at the plasma membrane with mp PKA-GFP. (C) Summary of all the experiments performed in the same conditions as in A and B. (D) Time to reach half-maximal response (t/2) to 10 μM PGE 1 . (E) The summary of experiments performed by applying 1 μM PGE 1 . Error bars indicate SEM. *, 0.01

    Article Snippet: Patch pipettes were filled with an intracellular solution, ICS-1, containing 120 mM K- aspartate, 10 mM TEA-Cl, 1 mM MgCl2 , 10 mM Hepes, 10 mM CsCl, 0.3 mM GTP-Na, 3 mM ATP-K (adjusted to pH 7.2 with KOH), 5 mM BAPTA, 1 mM thapsigargin, 0.1 mM IBMX, and 0.007–0.18 mM cAMP as indicated and filtered through 0.22-μm pores (Millipore).

    Techniques:

    A unimolecular Epac-based sensor detects different [cAMP] at the plasma membrane and in the bulk cytosol. (A) Schematic representation of the fusion protein constituting the Epac-based cAMP sensors H30 and confocal micrographs showing its distribution in HEK293 cells. (B) Structure and localization of the membrane-targeted version of mp H30. Bars, 10 μm. (C) cAMP dose-response curves measured as the percent FRET changes of H30 (white circles), mp H30 (black circles), and nls H30 (gray circles). EC 50 are 12.5, 20, and 17.5 μM, respectively. (D) Representative kinetics of cAMP changes generated in the cytosol (white circles) and at the plasma membrane (black circles) upon stimulation with 1 μM PGE 1 followed by 100 μM IBMX. (E) Summary of the experiments performed as in D. Error bars represent SEM. **, P = 0.002; ***, P = 10 −4 .

    Journal: The Journal of Cell Biology

    Article Title: PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases

    doi: 10.1083/jcb.200605050

    Figure Lengend Snippet: A unimolecular Epac-based sensor detects different [cAMP] at the plasma membrane and in the bulk cytosol. (A) Schematic representation of the fusion protein constituting the Epac-based cAMP sensors H30 and confocal micrographs showing its distribution in HEK293 cells. (B) Structure and localization of the membrane-targeted version of mp H30. Bars, 10 μm. (C) cAMP dose-response curves measured as the percent FRET changes of H30 (white circles), mp H30 (black circles), and nls H30 (gray circles). EC 50 are 12.5, 20, and 17.5 μM, respectively. (D) Representative kinetics of cAMP changes generated in the cytosol (white circles) and at the plasma membrane (black circles) upon stimulation with 1 μM PGE 1 followed by 100 μM IBMX. (E) Summary of the experiments performed as in D. Error bars represent SEM. **, P = 0.002; ***, P = 10 −4 .

    Article Snippet: Patch pipettes were filled with an intracellular solution, ICS-1, containing 120 mM K- aspartate, 10 mM TEA-Cl, 1 mM MgCl2 , 10 mM Hepes, 10 mM CsCl, 0.3 mM GTP-Na, 3 mM ATP-K (adjusted to pH 7.2 with KOH), 5 mM BAPTA, 1 mM thapsigargin, 0.1 mM IBMX, and 0.007–0.18 mM cAMP as indicated and filtered through 0.22-μm pores (Millipore).

    Techniques: Generated

    Role of PKA in shaping the cAMP gradient between the plasma membrane and the cytosol. (A and B) Representative cAMP kinetic ( Romoser et al., 1996 ) and summary of experiments (B) performed in HEK293 cells cotransfected with PKA and either H30 or mp H30 and challenged with 1 μM PGE 1 followed by either total PDE inhibition with 100 μM IBMX or PKA inhibition with 10 μM H89 as indicated. (C and D) Representative kinetics (C) and summary of experiments (D) showing the effect of endogenous PKA inhibition on the cAMP response induced by 10 nM PGE 1 at the plasma membrane and bulk cytosol in the absence and presence of the PKA inhibitor H89 (10 μM). In all of the experiments, when the PKA inhibitor was used, cells were preincubated for 10 min with H89, and the inhibitor was present throughout the experiment. Error bars represent SEM. *, P = 0.02; **, P = 0.009; ***, P = 0.0009.

    Journal: The Journal of Cell Biology

    Article Title: PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases

    doi: 10.1083/jcb.200605050

    Figure Lengend Snippet: Role of PKA in shaping the cAMP gradient between the plasma membrane and the cytosol. (A and B) Representative cAMP kinetic ( Romoser et al., 1996 ) and summary of experiments (B) performed in HEK293 cells cotransfected with PKA and either H30 or mp H30 and challenged with 1 μM PGE 1 followed by either total PDE inhibition with 100 μM IBMX or PKA inhibition with 10 μM H89 as indicated. (C and D) Representative kinetics (C) and summary of experiments (D) showing the effect of endogenous PKA inhibition on the cAMP response induced by 10 nM PGE 1 at the plasma membrane and bulk cytosol in the absence and presence of the PKA inhibitor H89 (10 μM). In all of the experiments, when the PKA inhibitor was used, cells were preincubated for 10 min with H89, and the inhibitor was present throughout the experiment. Error bars represent SEM. *, P = 0.02; **, P = 0.009; ***, P = 0.0009.

    Article Snippet: Patch pipettes were filled with an intracellular solution, ICS-1, containing 120 mM K- aspartate, 10 mM TEA-Cl, 1 mM MgCl2 , 10 mM Hepes, 10 mM CsCl, 0.3 mM GTP-Na, 3 mM ATP-K (adjusted to pH 7.2 with KOH), 5 mM BAPTA, 1 mM thapsigargin, 0.1 mM IBMX, and 0.007–0.18 mM cAMP as indicated and filtered through 0.22-μm pores (Millipore).

    Techniques: Inhibition

    Lentiviral transfer of Gsα R201C mutation in hBMSCs (A) LV vectors used for Gsα R201C transfer. LV-EF-Gsα R201C encodes the Gsα R201C protein (HA-tagged), controlled by the EF-1α promoter. The bidirectional LV vector (LV-BD-Gsα R201C ) encodes Gsα R201C and eGFP, under the control of the PGK and the CMV minimal promoters respectively. (B) Western blot analysis of Gsα R201C expression in LV-EF-Gsα R201C -transduced hBMSCs. Immunoblotting for HA demonstrates the specific signal for the mutated Gsα protein. Immunoblotting for Gsα demonstrates that mock-treated and empty vector (LV-Ctr)-transduced cells express comparable amounts of the 48KD and 43KD isoforms of Gsα (Gsα- long and Gsα- short , respectively) resulting from alternative splicing of exon 3. A specific increase of Gsα- long is observed in cells transduced with LV-EF-Gsα R201C . (C) Gsα R201C expression in hBMSCs is detected by immunofluorescence, as indicated by HA immunolabeling. (D) Intracellular cAMP levels were measured in control or LV-EF-Gsα R201C -transduced hBMSCs. In the presence of IBMX (1 mM) alone, or of IBMX and forskolin (10 μM), significantly higher levels of cAMP are observed in Gsα R201C expressing cells compared to control cells. Data from 5 separate experiments in duplicate are expressed as mean ± SD. a, p

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: Transfer, analysis and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors

    doi: 10.1359/jbmr.091036

    Figure Lengend Snippet: Lentiviral transfer of Gsα R201C mutation in hBMSCs (A) LV vectors used for Gsα R201C transfer. LV-EF-Gsα R201C encodes the Gsα R201C protein (HA-tagged), controlled by the EF-1α promoter. The bidirectional LV vector (LV-BD-Gsα R201C ) encodes Gsα R201C and eGFP, under the control of the PGK and the CMV minimal promoters respectively. (B) Western blot analysis of Gsα R201C expression in LV-EF-Gsα R201C -transduced hBMSCs. Immunoblotting for HA demonstrates the specific signal for the mutated Gsα protein. Immunoblotting for Gsα demonstrates that mock-treated and empty vector (LV-Ctr)-transduced cells express comparable amounts of the 48KD and 43KD isoforms of Gsα (Gsα- long and Gsα- short , respectively) resulting from alternative splicing of exon 3. A specific increase of Gsα- long is observed in cells transduced with LV-EF-Gsα R201C . (C) Gsα R201C expression in hBMSCs is detected by immunofluorescence, as indicated by HA immunolabeling. (D) Intracellular cAMP levels were measured in control or LV-EF-Gsα R201C -transduced hBMSCs. In the presence of IBMX (1 mM) alone, or of IBMX and forskolin (10 μM), significantly higher levels of cAMP are observed in Gsα R201C expressing cells compared to control cells. Data from 5 separate experiments in duplicate are expressed as mean ± SD. a, p

    Article Snippet: Cells were cultured for two weeks, then plated in 48-well plates at a density of 15,000 cells/well and incubated for 1 hr in serum-free medium in the presence or absence of 1 mM IBMX (Sigma) or 1 mM IBMX and 10 μM forskolin (Calbiochem). cAMP was measured using the cAMP Direct Biotrak™ EIA kit (GE Healthcare) according to manufacturer’s instructions. ( )

    Techniques: Mutagenesis, Plasmid Preparation, Western Blot, Expressing, Transduction, Immunofluorescence, Immunolabeling

    Activity, mRNA expression and phenotypic effects of PDEs in mutation-transduced hBMSCs (A) PDE activity was assayed in hBMSCs, mock-treated or transduced with LV-Ctr or LV- EF-Gsα R201C , using 10 μM of cAMP as a substrate, in the presence or absence of either IBMX (1 mM) or Rolipram (20 μM). b, p

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: Transfer, analysis and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors

    doi: 10.1359/jbmr.091036

    Figure Lengend Snippet: Activity, mRNA expression and phenotypic effects of PDEs in mutation-transduced hBMSCs (A) PDE activity was assayed in hBMSCs, mock-treated or transduced with LV-Ctr or LV- EF-Gsα R201C , using 10 μM of cAMP as a substrate, in the presence or absence of either IBMX (1 mM) or Rolipram (20 μM). b, p

    Article Snippet: Cells were cultured for two weeks, then plated in 48-well plates at a density of 15,000 cells/well and incubated for 1 hr in serum-free medium in the presence or absence of 1 mM IBMX (Sigma) or 1 mM IBMX and 10 μM forskolin (Calbiochem). cAMP was measured using the cAMP Direct Biotrak™ EIA kit (GE Healthcare) according to manufacturer’s instructions. ( )

    Techniques: Activity Assay, Expressing, Mutagenesis, Transduction

    Metformin-induced secretion in human L cells. ( A ) Colonic epithelial preparations readily secrete glucagon-like peptide 1 (GLP-1) in response to high (70 mM) external K + or to a combination of 3-isobutyl-1-methylxanthine (IBMX) and forskolin (FSK) ( n = 22). ( B and C ) Metformin (10 μM) increases GLP-1 release after 15 minutes in epithelial tissue from human colon ( n = 46) ( B ) and ileum ( n = 10) ( C ). ( D ) Metformin-induced GLP-1 release is blocked by the AMP-activated protein kinase (AMPK) inhibitor dorsomorphin ( n = 18). ( E ) Metformin-induced GLP-1 release is blocked by the serotonin transporter (SERT) inhibitor fluoxetine and the plasma membrane monoamine transporter (PMAT) lopinavir ( n = 18). Bar graph data are means ± SEM. * P

    Journal: JCI Insight

    Article Title: Metformin-induced glucagon-like peptide-1 secretion contributes to the actions of metformin in type 2 diabetes

    doi: 10.1172/jci.insight.93936

    Figure Lengend Snippet: Metformin-induced secretion in human L cells. ( A ) Colonic epithelial preparations readily secrete glucagon-like peptide 1 (GLP-1) in response to high (70 mM) external K + or to a combination of 3-isobutyl-1-methylxanthine (IBMX) and forskolin (FSK) ( n = 22). ( B and C ) Metformin (10 μM) increases GLP-1 release after 15 minutes in epithelial tissue from human colon ( n = 46) ( B ) and ileum ( n = 10) ( C ). ( D ) Metformin-induced GLP-1 release is blocked by the AMP-activated protein kinase (AMPK) inhibitor dorsomorphin ( n = 18). ( E ) Metformin-induced GLP-1 release is blocked by the serotonin transporter (SERT) inhibitor fluoxetine and the plasma membrane monoamine transporter (PMAT) lopinavir ( n = 18). Bar graph data are means ± SEM. * P

    Article Snippet: IBMX and FSK (I5879 and F6886, MilliporeSigma) (10 μM each) and 70 mM KCl were used as positive controls.

    Techniques:

    Change of Pαβγ's characteristics by cGMP binding. A . Dissociation of [ 3 H]cGMP bound to Pαβγ. Purified Pαβγ (16.0 μg) suspended in 640 μl of 55.5 mM Tris·HCl (pH 7.5) containing 4.44 mM EDTA and 1.11 mM IBMX, and [ 3 H]cGMP binding was initiated by adding 80 μl of 9 μM [ 3 H]cGMP. After incubation for 30 min on ice, an aliquot (72 μl) was withdrawn and applied to a Millipore filter, and its radioactivity was designated as the level at time 0. Simultaneously, 72 μl of 10 mM unlabeled cGMP (●) or water (○) was added to the assay mixture. After incubation for 0.25, 0.5, 0.75, 1, 2, 5, 10, and 20 min, an aliquot (80 μl) was withdrawn and applied to a Millipore filter, and its [ 3 H]-radioactivity was measured. The arrow indicates the addition of cGMP or water. The 100% activity indicates that 1.32 pmol of [ 3 H]cGMP was detected in 1.6 μg of Pαβγ (7.72 pmol). B . Elution profile of Pαβγ from a gel filtration column. Purified Pαβγ (70 μg) was incubated with (black) or without (red) unlabeled cGMP (0.5 mM) in 0.5 ml of 25 mM Tris·HCl, pH 7.5, 0.1 mM EDTA, and 1 mM IBMX for 30 min on ice and applied to a Superdex 200 HR column that had been equilibrated with Buffer E. Detailed conditions for this elution are in Experimental Procedure. PDE activity was assayed using 5 μl of the fraction (●). The 100% PDE activity indicates that 12.5 nmol cGMP hydrolyzed/min/tube. [ 3 H]cGMP binding activity was measured using 50 μl of the fraction (□). The 100% activity indicates that 1.50 pmol of [ 3 H]cGMP was detected in the assay mixture.

    Journal: The FEBS journal

    Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

    doi: 10.1111/j.1742-4658.2011.08104.x

    Figure Lengend Snippet: Change of Pαβγ's characteristics by cGMP binding. A . Dissociation of [ 3 H]cGMP bound to Pαβγ. Purified Pαβγ (16.0 μg) suspended in 640 μl of 55.5 mM Tris·HCl (pH 7.5) containing 4.44 mM EDTA and 1.11 mM IBMX, and [ 3 H]cGMP binding was initiated by adding 80 μl of 9 μM [ 3 H]cGMP. After incubation for 30 min on ice, an aliquot (72 μl) was withdrawn and applied to a Millipore filter, and its radioactivity was designated as the level at time 0. Simultaneously, 72 μl of 10 mM unlabeled cGMP (●) or water (○) was added to the assay mixture. After incubation for 0.25, 0.5, 0.75, 1, 2, 5, 10, and 20 min, an aliquot (80 μl) was withdrawn and applied to a Millipore filter, and its [ 3 H]-radioactivity was measured. The arrow indicates the addition of cGMP or water. The 100% activity indicates that 1.32 pmol of [ 3 H]cGMP was detected in 1.6 μg of Pαβγ (7.72 pmol). B . Elution profile of Pαβγ from a gel filtration column. Purified Pαβγ (70 μg) was incubated with (black) or without (red) unlabeled cGMP (0.5 mM) in 0.5 ml of 25 mM Tris·HCl, pH 7.5, 0.1 mM EDTA, and 1 mM IBMX for 30 min on ice and applied to a Superdex 200 HR column that had been equilibrated with Buffer E. Detailed conditions for this elution are in Experimental Procedure. PDE activity was assayed using 5 μl of the fraction (●). The 100% PDE activity indicates that 12.5 nmol cGMP hydrolyzed/min/tube. [ 3 H]cGMP binding activity was measured using 50 μl of the fraction (□). The 100% activity indicates that 1.50 pmol of [ 3 H]cGMP was detected in the assay mixture.

    Article Snippet: Typically, Pαβγ (∼15 μg before purification, and ∼2 μg, after purification) was incubated with 0.5 or 1 μM [3 H]cGMP (1 mCi/ml) in the assay medium (final volume, 100 μl) containing 25 mM Tris·HCl, pH 7.5, 0.1 mM EDTA, and 1 mM IBMX for 30 min on ice, and 80 μl aliquots were applied to a Millipore filter (HA, pore size 0.45 μm) that had been wetted with 20 mM sodium phosphate buffer (pH 6.5).

    Techniques: Binding Assay, Purification, Incubation, Radioactivity, Activity Assay, Filtration

    Binding of [ 3 H]cGMP to Pαβγ. A . Concentration of [ 3 H]cGMP. [ 3 H]cGMP binding to Pαβγ (1.92 μg) was measured with indicated concentrations of [ 3 H]cGMP. The [ 3 H]cGMP-binding activity was analyzed by Scatchard plotting (Insert). B . Time-course. Pαβγ (17.3 μg) was incubated in 55 mM Tris·HCl, (pH 7.5) containing 4.4 mM EDTA and 1.1 mM IBMX (final volume, 720 μl) on ice for 10 min. The [ 3 H]cGMP binding was initiated by adding 80 μl of 10 μM [ 3 H]cGMP. After incubation for indicated periods, an aliquot (80 μl) was taken and applied to a Millipore filter. C . The cyclic nucleotide-specificity. After incubation of Pαβγ (1.92 μg) with indicated concentration of unlabeled cGMP (●) or cAMP (○) on ice for 10 min, [ 3 H]cGMP binding was measured with 1 μM [ 3 H]cGMP. The 100% activity indicates that 1.46 pmol [ 3 H]cGMP bound to Pαβγ in tubes. D . Levels of [ 3 H]cGMP-bound Pαβγ trapped by the filter. OS homogenates (18.9 mg protein) were suspended in 9.7 ml of Buffer A. After isolation by the TSK-DEAE 5PW column chromatography and concentration to 0.3 ml, the Pαβγ preparation (∼80 μg) was incubated with 1 μM [ 3 H]cGMP for 30 min on ice and applied to a TSK 250 column that had been equilibrated with Buffer D. The level of [ 3 H]cGMP bound to Pαβγ was calculated based on the [ 3 H]-radioactivity in 70 μl of the fraction (●). The fraction (70 μl) was also applied to a Millipore filter and the [ 3 H]-radioactivity on the filter was measured (□). Only fractions containing Pαβγ were shown. Insert: The rate of [ 3 H]-radioactivity on the filter per the level of [ 3 H]-radioactivity in the fraction. The 100% radioactivity indicates the [ 3 H]-radioactivity detected in fraction 15. Fraction 15 (70 μl) contained 3.2 μg Pαβγ (15.5 pmol) and 13.1 pmol of [ 3 H]cGMP.

    Journal: The FEBS journal

    Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

    doi: 10.1111/j.1742-4658.2011.08104.x

    Figure Lengend Snippet: Binding of [ 3 H]cGMP to Pαβγ. A . Concentration of [ 3 H]cGMP. [ 3 H]cGMP binding to Pαβγ (1.92 μg) was measured with indicated concentrations of [ 3 H]cGMP. The [ 3 H]cGMP-binding activity was analyzed by Scatchard plotting (Insert). B . Time-course. Pαβγ (17.3 μg) was incubated in 55 mM Tris·HCl, (pH 7.5) containing 4.4 mM EDTA and 1.1 mM IBMX (final volume, 720 μl) on ice for 10 min. The [ 3 H]cGMP binding was initiated by adding 80 μl of 10 μM [ 3 H]cGMP. After incubation for indicated periods, an aliquot (80 μl) was taken and applied to a Millipore filter. C . The cyclic nucleotide-specificity. After incubation of Pαβγ (1.92 μg) with indicated concentration of unlabeled cGMP (●) or cAMP (○) on ice for 10 min, [ 3 H]cGMP binding was measured with 1 μM [ 3 H]cGMP. The 100% activity indicates that 1.46 pmol [ 3 H]cGMP bound to Pαβγ in tubes. D . Levels of [ 3 H]cGMP-bound Pαβγ trapped by the filter. OS homogenates (18.9 mg protein) were suspended in 9.7 ml of Buffer A. After isolation by the TSK-DEAE 5PW column chromatography and concentration to 0.3 ml, the Pαβγ preparation (∼80 μg) was incubated with 1 μM [ 3 H]cGMP for 30 min on ice and applied to a TSK 250 column that had been equilibrated with Buffer D. The level of [ 3 H]cGMP bound to Pαβγ was calculated based on the [ 3 H]-radioactivity in 70 μl of the fraction (●). The fraction (70 μl) was also applied to a Millipore filter and the [ 3 H]-radioactivity on the filter was measured (□). Only fractions containing Pαβγ were shown. Insert: The rate of [ 3 H]-radioactivity on the filter per the level of [ 3 H]-radioactivity in the fraction. The 100% radioactivity indicates the [ 3 H]-radioactivity detected in fraction 15. Fraction 15 (70 μl) contained 3.2 μg Pαβγ (15.5 pmol) and 13.1 pmol of [ 3 H]cGMP.

    Article Snippet: Typically, Pαβγ (∼15 μg before purification, and ∼2 μg, after purification) was incubated with 0.5 or 1 μM [3 H]cGMP (1 mCi/ml) in the assay medium (final volume, 100 μl) containing 25 mM Tris·HCl, pH 7.5, 0.1 mM EDTA, and 1 mM IBMX for 30 min on ice, and 80 μl aliquots were applied to a Millipore filter (HA, pore size 0.45 μm) that had been wetted with 20 mM sodium phosphate buffer (pH 6.5).

    Techniques: Binding Assay, Concentration Assay, Activity Assay, Incubation, Isolation, Column Chromatography, Radioactivity

    Effects of Pγ and its mutants on [ 3 H]cGMP binding to Pαβγ. A . The effect on the level of [ 3 H]cGMP binding. After incubation or Pαβγ (1.92 μg) with various concentrations of Pγ or its mutants, the [ 3 H]cGMP-binding activity was measured. The 100% activity indicates that 1.46 pmol [ 3 H]cGMP bound to Pαβγ in tubes. Following Pγ and its mutants were used: ●, wild type Pγ; □, N18del; △, N22del; ▲, C18Sub, and ▼, C10del. B . The effect on the time-course of [ 3 H]cGMP-binding. Pαβγ (17.3 μg) was incubated with 1.11 μM Pγ or its mutants in 55 mM Tris·HCl, (pH 7.5) containing 4.4 mM EDTA and 1.1 mM IBMX (final volume, 720 μl) on ice for 30 min. The [ 3 H]cGMP binding was initiated by adding 80 μl of 10 μM [ 3 H]cGMP. After incubation for indicated periods on ice, an aliquot (80 μl) was taken and applied to a Millipore filter. Following Pγ and its mutants were used: ○, control; ●, wild type Pγ; △, N22del; and ▲, C18Sub. C . The effect on the Scatchard plot. Pαβγ (1.92 μg) was incubated with 1 mM of wild type Pγ (●) or N22del (△). As a control, Pαβγ alone was incubated (○). Then, [ 3 H]cGMP binding was initiated by adding indicated concentrations of [ 3 H]cGMP ( C-1 ). The [ 3 H]cGMP binding in C-1 is analyzed by Scatchard plotting ( C-2 ).

    Journal: The FEBS journal

    Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

    doi: 10.1111/j.1742-4658.2011.08104.x

    Figure Lengend Snippet: Effects of Pγ and its mutants on [ 3 H]cGMP binding to Pαβγ. A . The effect on the level of [ 3 H]cGMP binding. After incubation or Pαβγ (1.92 μg) with various concentrations of Pγ or its mutants, the [ 3 H]cGMP-binding activity was measured. The 100% activity indicates that 1.46 pmol [ 3 H]cGMP bound to Pαβγ in tubes. Following Pγ and its mutants were used: ●, wild type Pγ; □, N18del; △, N22del; ▲, C18Sub, and ▼, C10del. B . The effect on the time-course of [ 3 H]cGMP-binding. Pαβγ (17.3 μg) was incubated with 1.11 μM Pγ or its mutants in 55 mM Tris·HCl, (pH 7.5) containing 4.4 mM EDTA and 1.1 mM IBMX (final volume, 720 μl) on ice for 30 min. The [ 3 H]cGMP binding was initiated by adding 80 μl of 10 μM [ 3 H]cGMP. After incubation for indicated periods on ice, an aliquot (80 μl) was taken and applied to a Millipore filter. Following Pγ and its mutants were used: ○, control; ●, wild type Pγ; △, N22del; and ▲, C18Sub. C . The effect on the Scatchard plot. Pαβγ (1.92 μg) was incubated with 1 mM of wild type Pγ (●) or N22del (△). As a control, Pαβγ alone was incubated (○). Then, [ 3 H]cGMP binding was initiated by adding indicated concentrations of [ 3 H]cGMP ( C-1 ). The [ 3 H]cGMP binding in C-1 is analyzed by Scatchard plotting ( C-2 ).

    Article Snippet: Typically, Pαβγ (∼15 μg before purification, and ∼2 μg, after purification) was incubated with 0.5 or 1 μM [3 H]cGMP (1 mCi/ml) in the assay medium (final volume, 100 μl) containing 25 mM Tris·HCl, pH 7.5, 0.1 mM EDTA, and 1 mM IBMX for 30 min on ice, and 80 μl aliquots were applied to a Millipore filter (HA, pore size 0.45 μm) that had been wetted with 20 mM sodium phosphate buffer (pH 6.5).

    Techniques: Binding Assay, Incubation, Activity Assay