forskolin  (Alomone Labs)


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    Structured Review

    Alomone Labs forskolin
    Neurite outgrowth is regulated by cAMP signaling. A. Plated neurospheres were treated with 10 μM <t>forskolin</t> for 24 h. The number of neurites crossing a circular mask around the neurosphere was used for quantification. B. Micrographs of control and 10 μM forskolin-treated neurospheres immunostained for beta III tubulin (green). Nuclei counterstained with DAPI (blue). C. Number of neurites crossing the circle (**p
    Forskolin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/forskolin/product/Alomone Labs
    Average 93 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    forskolin - by Bioz Stars, 2022-08
    93/100 stars

    Images

    1) Product Images from "Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres"

    Article Title: Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres

    Journal: bioRxiv

    doi: 10.1101/2021.02.21.432144

    Neurite outgrowth is regulated by cAMP signaling. A. Plated neurospheres were treated with 10 μM forskolin for 24 h. The number of neurites crossing a circular mask around the neurosphere was used for quantification. B. Micrographs of control and 10 μM forskolin-treated neurospheres immunostained for beta III tubulin (green). Nuclei counterstained with DAPI (blue). C. Number of neurites crossing the circle (**p
    Figure Legend Snippet: Neurite outgrowth is regulated by cAMP signaling. A. Plated neurospheres were treated with 10 μM forskolin for 24 h. The number of neurites crossing a circular mask around the neurosphere was used for quantification. B. Micrographs of control and 10 μM forskolin-treated neurospheres immunostained for beta III tubulin (green). Nuclei counterstained with DAPI (blue). C. Number of neurites crossing the circle (**p

    Techniques Used:

    2) Product Images from "Regulation of basal expression of hepatic PEPCK and G6Pase by AKT2"

    Article Title: Regulation of basal expression of hepatic PEPCK and G6Pase by AKT2

    Journal: The Biochemical journal

    doi: 10.1042/BCJ20190570

    CREB phosphorylation mediates AKT2-regulated induction of PEPCK. ( A ) CREB phosphorylation induced by PTEN loss is regulated by AKT2. Liver lysates from mice with different genotypes were analysis for p-CREB. PM, Pten deleted; DM, Pten and Akt2 deleted; AM, Akt2 deleted. Two biological replicas are included for each genotype group. Protein levels of p-AKT, PTEN and p-GSK3 are analyzed and showed up-regulation of AKT and GSK-3 with PTEN loss and inhibition when AKT2 is lost. Phosphorylation of CREB is only observed when PTEN is lost. AKT2 loss together with PTEN blocked this phosphorylation in the DM livers. ( B ) Akt2 +/+ and Akt2 − / − cell were treated with forskolin (100 μM) for 24 h to induce cAMP and CREB phosphorylation or vehicle as controls. Phosphorylation of CREB is detected and showed to be lower when AKT2 is lost. When forskolin is added to induce cAMP, protein expression of p-CREB is evaluated in both Akt2 +/+ and Akt2 − / − cells even though its levels remains lower in the Akt2 − / − cells. Vinculin is probed as loading control. ( C ) Introduction of CREB-133A and wtCREB to Akt2 +/+ and Akt2 −/− hepatocytes. Expression of PEPCK is analyzed using quantitative PCR analysis. Left panel, CREB-S133A is introduced to Akt2 +/+ cells. Middle panel, wtCREB is introduced to Akt2 −/− cells; Right panel, wtCREB is introduced to Akt2 +/+ cells. n = 3. Data presented as mean ± SEM. * P ≤ 0.05. Different from vector transfected cells of the same genotype. Experiments repeated at least three times.
    Figure Legend Snippet: CREB phosphorylation mediates AKT2-regulated induction of PEPCK. ( A ) CREB phosphorylation induced by PTEN loss is regulated by AKT2. Liver lysates from mice with different genotypes were analysis for p-CREB. PM, Pten deleted; DM, Pten and Akt2 deleted; AM, Akt2 deleted. Two biological replicas are included for each genotype group. Protein levels of p-AKT, PTEN and p-GSK3 are analyzed and showed up-regulation of AKT and GSK-3 with PTEN loss and inhibition when AKT2 is lost. Phosphorylation of CREB is only observed when PTEN is lost. AKT2 loss together with PTEN blocked this phosphorylation in the DM livers. ( B ) Akt2 +/+ and Akt2 − / − cell were treated with forskolin (100 μM) for 24 h to induce cAMP and CREB phosphorylation or vehicle as controls. Phosphorylation of CREB is detected and showed to be lower when AKT2 is lost. When forskolin is added to induce cAMP, protein expression of p-CREB is evaluated in both Akt2 +/+ and Akt2 − / − cells even though its levels remains lower in the Akt2 − / − cells. Vinculin is probed as loading control. ( C ) Introduction of CREB-133A and wtCREB to Akt2 +/+ and Akt2 −/− hepatocytes. Expression of PEPCK is analyzed using quantitative PCR analysis. Left panel, CREB-S133A is introduced to Akt2 +/+ cells. Middle panel, wtCREB is introduced to Akt2 −/− cells; Right panel, wtCREB is introduced to Akt2 +/+ cells. n = 3. Data presented as mean ± SEM. * P ≤ 0.05. Different from vector transfected cells of the same genotype. Experiments repeated at least three times.

    Techniques Used: Mouse Assay, Inhibition, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection

    3) Product Images from "Functional and Molecular Evidence for Kv7 Channel Subtypes in Human Detrusor from Patients with and without Bladder Outflow Obstruction"

    Article Title: Functional and Molecular Evidence for Kv7 Channel Subtypes in Human Detrusor from Patients with and without Bladder Outflow Obstruction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0117350

    At low depolarisation K v 7 channel inhibition reduces the response to forskolin. Strips pre-constricted with 20 mM KPSS were treated with either 0.1 μM isoprenaline (A) or 1 μM forskolin (B). Pre-treatment with K v 7 channel modulators revealed a reduction in response to isoprenaline and forskolin when blocking K v 7 channels by XE991 (10 μM) whereas retigabine (10μM) did not affect the response to isoprenaline. Pre-treatment with vehicle control (0.1% DMSO). ** P
    Figure Legend Snippet: At low depolarisation K v 7 channel inhibition reduces the response to forskolin. Strips pre-constricted with 20 mM KPSS were treated with either 0.1 μM isoprenaline (A) or 1 μM forskolin (B). Pre-treatment with K v 7 channel modulators revealed a reduction in response to isoprenaline and forskolin when blocking K v 7 channels by XE991 (10 μM) whereas retigabine (10μM) did not affect the response to isoprenaline. Pre-treatment with vehicle control (0.1% DMSO). ** P

    Techniques Used: Inhibition, Blocking Assay

    Modulation of K v 7 channels does not affect Gs mediated relaxation at 1 μM carbachol-induced contractility. Cumulative concentrations-responses for isoprenaline (A and B) and forskolin (C) were produced with pre-incubation of 10 μM XE991 (A and C), 10 μM retigabine (B) or vehicle control (0.1% DMSO). Initial tone was achieved by pre-constriction with 1 μM carbachol. * P
    Figure Legend Snippet: Modulation of K v 7 channels does not affect Gs mediated relaxation at 1 μM carbachol-induced contractility. Cumulative concentrations-responses for isoprenaline (A and B) and forskolin (C) were produced with pre-incubation of 10 μM XE991 (A and C), 10 μM retigabine (B) or vehicle control (0.1% DMSO). Initial tone was achieved by pre-constriction with 1 μM carbachol. * P

    Techniques Used: Produced, Incubation

    Modulation of K v 7 channels does not affect Gs mediated relaxation at 40 mM KPSS-induced contractility. Cumulative concentrations-responses for isoprenaline (A and B) and forskolin (C) were produced with pre-incubation of 10 μM XE991 (A and C), 10 μM retigabine (B) or vehicle control (0.1% DMSO). Initial tone was achieved by pre-constriction with 40 mM KPSS. Neither of the K v 7 channel modulators affected the response to isoprenaline or forskolin.
    Figure Legend Snippet: Modulation of K v 7 channels does not affect Gs mediated relaxation at 40 mM KPSS-induced contractility. Cumulative concentrations-responses for isoprenaline (A and B) and forskolin (C) were produced with pre-incubation of 10 μM XE991 (A and C), 10 μM retigabine (B) or vehicle control (0.1% DMSO). Initial tone was achieved by pre-constriction with 40 mM KPSS. Neither of the K v 7 channel modulators affected the response to isoprenaline or forskolin.

    Techniques Used: Produced, Incubation

    Effect of isoprenaline and forskolin in human DSM at 1 μM carbachol- and 40 mM KPSS-induced contractions. Cumulative concentration-responses were induced with isoprenaline and forskolin. Initial tone was achieved by pre-constriction with 1 μM carbachol (A and B) and 40 mM KPSS (C and D) bladder strips. * P
    Figure Legend Snippet: Effect of isoprenaline and forskolin in human DSM at 1 μM carbachol- and 40 mM KPSS-induced contractions. Cumulative concentration-responses were induced with isoprenaline and forskolin. Initial tone was achieved by pre-constriction with 1 μM carbachol (A and B) and 40 mM KPSS (C and D) bladder strips. * P

    Techniques Used: Concentration Assay

    4) Product Images from "Caveolin-1 and Lipid Microdomains Regulate Gs Trafficking and Attenuate Gs/Adenylyl Cyclase Signaling"

    Article Title: Caveolin-1 and Lipid Microdomains Regulate Gs Trafficking and Attenuate Gs/Adenylyl Cyclase Signaling

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.109.060160

    Caveolin knockdown increases forskolin- and fluoride-stimulated Gα s /adenylyl cyclase signaling. A, wild-type C6 cells (WT C6) or caveolin-1 stable knockdown cells (CAV-1 RNAi) were incubated with [2,8- 3 H]adenine for 18 h to label cellular ATP.
    Figure Legend Snippet: Caveolin knockdown increases forskolin- and fluoride-stimulated Gα s /adenylyl cyclase signaling. A, wild-type C6 cells (WT C6) or caveolin-1 stable knockdown cells (CAV-1 RNAi) were incubated with [2,8- 3 H]adenine for 18 h to label cellular ATP.

    Techniques Used: Incubation

    Caveolin-1 knockout increases fluoride and forskolin-stimulated adenylyl cyclase activity in mouse striatum. Ten- to 12-week-old wild-type or caveolin-1 knockout littermate mice were colony bred, brains were obtained, and striatum was microdissected.
    Figure Legend Snippet: Caveolin-1 knockout increases fluoride and forskolin-stimulated adenylyl cyclase activity in mouse striatum. Ten- to 12-week-old wild-type or caveolin-1 knockout littermate mice were colony bred, brains were obtained, and striatum was microdissected.

    Techniques Used: Knock-Out, Activity Assay, Mouse Assay

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    Alomone Labs p2x7 intracellular epitope antibody
    <t>P2X7</t> receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.
    P2x7 Intracellular Epitope Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2x7 intracellular epitope antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p2x7 intracellular epitope antibody - by Bioz Stars, 2022-08
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    85
    Alomone Labs anti α actin
    Kv7.1 and Kv7.5 expression in optimal cutting temperature compound (OCT)-embedded malignant vascular tumors. Consecutive sections of vascular tumor samples were stained by hematoxylin-eosin (HE) and immunofluorescent labeling for Kv7.1 and Kv7.5. ( Aa – Ac ) Cutaneous angiosarcoma. ( Ba – Bc ) Epithelioid haemangioendothelioma. ( Ca – Cc ) Testicular teratoma-derived angiosarcoma. ( Aa , Ba , Ca ) HE was used to identify structures. BV: healthy blood vessels; *: tumoral blood vessels. Scale bars 150 μm. Representative immunolabeling images showing merged Kv7.1 ( Ab , Bb , Cb ) and Kv7.5 ( Ac , Bc , Cc ) and tumor biopsies. Red, channels; green, <t>α-actin</t> (muscle); blue, DAPI nuclear staining. Scale bars 150 μm. ( D ) Tumor-to-normal (TN) ratios, in arbitrary units, were calculated from the protein expression levels obtained in immunofluorescence assays and represented in a forest plot. The analysis was performed for Kv7.1 ( top ) and Kv7.5 ( bottom ) channels, plotted in horizontal bars. The reference value (1) is indicated by a dotted line. Squares represent the mean ± SEM of the TN ratio for each patient and tumor. Gray, cutaneous angiosarcoma. White, epithelioid haemangioendothelioma. Black, testicular teratoma-derived angiosarcoma. Mean ± SEM, only represented when multiple samples and regions of interest (ROIs) were available.
    Anti α Actin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Alomone Labs forskolin
    Neurite outgrowth is regulated by cAMP signaling. A. Plated neurospheres were treated with 10 μM <t>forskolin</t> for 24 h. The number of neurites crossing a circular mask around the neurosphere was used for quantification. B. Micrographs of control and 10 μM forskolin-treated neurospheres immunostained for beta III tubulin (green). Nuclei counterstained with DAPI (blue). C. Number of neurites crossing the circle (**p
    Forskolin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/forskolin/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    forskolin - by Bioz Stars, 2022-08
    93/100 stars
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    Image Search Results


    P2X7 receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Expressing, Immunofluorescence

    Monocyte chemoattractant protein-1 (MCP-1) chemokine colocalizes with P2X7 receptor (P2X7R) on astrocytes in secondary progressive multiple sclerosis (SPMS) frontal cortex. Triple immunofluorescence confocal analysis performed on sections of frontal cortex from control (A–D) and SPMS (E–H) with antibodies for P2X7R [ (A,E) , red], MCP-1 [ (B,F) , green], and glial fibrillary acidic protein [ (C,G) , blue] shows colocalization [ (D,H) , white signal] and strong up-regulation of signals in white matter of SPMS (E–H) .

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: Monocyte chemoattractant protein-1 (MCP-1) chemokine colocalizes with P2X7 receptor (P2X7R) on astrocytes in secondary progressive multiple sclerosis (SPMS) frontal cortex. Triple immunofluorescence confocal analysis performed on sections of frontal cortex from control (A–D) and SPMS (E–H) with antibodies for P2X7R [ (A,E) , red], MCP-1 [ (B,F) , green], and glial fibrillary acidic protein [ (C,G) , blue] shows colocalization [ (D,H) , white signal] and strong up-regulation of signals in white matter of SPMS (E–H) .

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Immunofluorescence

    Spatiotemporal profile of P2X7 receptor (P2X7R) expression in multiple sclerosis (MS). The cartoon describes that P2X7R is down-regulated in monocytes during their activation and extravasation from blood vessel to MS cerebral cortex, while the receptor and the monocyte chemoattractant protein-1 chemokine are up-regulated on MS astrocytes concurrently with increased glial fibrillary acidic protein signal and glial scar formation.

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: Spatiotemporal profile of P2X7 receptor (P2X7R) expression in multiple sclerosis (MS). The cartoon describes that P2X7R is down-regulated in monocytes during their activation and extravasation from blood vessel to MS cerebral cortex, while the receptor and the monocyte chemoattractant protein-1 chemokine are up-regulated on MS astrocytes concurrently with increased glial fibrillary acidic protein signal and glial scar formation.

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Expressing, Activation Assay

    P2X7 receptor (P2X7R) is present on monocytes in blood vessels of secondary progressive multiple sclerosis (SPMS) frontal cortex. (A) Immunohistochemistry on sections from human frontal cortex shows roundish P2X7R-positive cells distributed within distinct clusters throughout the entire SPMS tissue. Confocal double immunofluorescence analysis of these clusters exhibits colocalization of P2X7R protein (red) with CD45 leukocyte marker [ (B) , green]. Staining with Lectin from Lycopersicon esculentum for vascular endothelial cells [ (C,D) , green] clearly shows the presence of P2X7R-positive roundish cells (red) within the lumen of blood vessels (asterisk). Double immunofluorescence of P2X7R-positive clusters shows colocalization of P2X7R (red) with cluster of differentiation 14 (CD14) monocyte/macrophage marker [ (E) , green]. Confocal triple immunofluorescence analysis is then performed with antibodies for CD14 [ (F,G) , green], P2X7R [ (F,G) , red], and microglia/macrophages markers CD68 (F) or major histocompatibility complex II [ (G) , blue]. The asterisk always indicates the lumen of blood vessels, arrows indicate P2X7R-negative cells, and arrowheads P2X7R-positive cells.

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) is present on monocytes in blood vessels of secondary progressive multiple sclerosis (SPMS) frontal cortex. (A) Immunohistochemistry on sections from human frontal cortex shows roundish P2X7R-positive cells distributed within distinct clusters throughout the entire SPMS tissue. Confocal double immunofluorescence analysis of these clusters exhibits colocalization of P2X7R protein (red) with CD45 leukocyte marker [ (B) , green]. Staining with Lectin from Lycopersicon esculentum for vascular endothelial cells [ (C,D) , green] clearly shows the presence of P2X7R-positive roundish cells (red) within the lumen of blood vessels (asterisk). Double immunofluorescence of P2X7R-positive clusters shows colocalization of P2X7R (red) with cluster of differentiation 14 (CD14) monocyte/macrophage marker [ (E) , green]. Confocal triple immunofluorescence analysis is then performed with antibodies for CD14 [ (F,G) , green], P2X7R [ (F,G) , red], and microglia/macrophages markers CD68 (F) or major histocompatibility complex II [ (G) , blue]. The asterisk always indicates the lumen of blood vessels, arrows indicate P2X7R-negative cells, and arrowheads P2X7R-positive cells.

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Immunohistochemistry, Immunofluorescence, Marker, Staining

    P2X7 receptor (P2X7R) is present on astrocytes in the parenchyma of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal analysis of SPMS frontal cortex parenchyma shows absence of colocalization of P2X7R (red) with P2Y12R [ (A) , green], major histocompatibility complex II (MHC II) [ (B) , blue], and myelin basic protein [ (C) , blue], but the presence of merged P2X7R/glial fibrillary acidic protein signals (D–F) . P2X7R/MHC II-positive signal is also visible but confined in the lumen of a blood vessel [ (B) , arrow, pink]. Immunohistochemistry analysis of cortical parenchyma reveals the abundant presence of P2X7R in GM on interlaminar (G) and protoplasmic astrocytes (E) , and in white matter on fibrous astrocytes (I,J) . In (J) , astrocytic “vascular feet” are visible adjacent to the blood vessel walls (arrows).

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) is present on astrocytes in the parenchyma of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal analysis of SPMS frontal cortex parenchyma shows absence of colocalization of P2X7R (red) with P2Y12R [ (A) , green], major histocompatibility complex II (MHC II) [ (B) , blue], and myelin basic protein [ (C) , blue], but the presence of merged P2X7R/glial fibrillary acidic protein signals (D–F) . P2X7R/MHC II-positive signal is also visible but confined in the lumen of a blood vessel [ (B) , arrow, pink]. Immunohistochemistry analysis of cortical parenchyma reveals the abundant presence of P2X7R in GM on interlaminar (G) and protoplasmic astrocytes (E) , and in white matter on fibrous astrocytes (I,J) . In (J) , astrocytic “vascular feet” are visible adjacent to the blood vessel walls (arrows).

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Immunohistochemistry

    P2X7 receptor (P2X7R) immunoreactivity is increased with astrogliosis in white matter (WM) of secondary progressive multiple sclerosis (SPMS) frontal cortex parenchyma. Immunohistochemistry analysis of two adjacent WM fields from SPMS frontal cortex characterized, respectively, by hypertrophic astrocytes (B) and glial scar (C) reveals a noteworthy increase in P2X7R-positive astrocytes (B,C) , compared with control case (A) .

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) immunoreactivity is increased with astrogliosis in white matter (WM) of secondary progressive multiple sclerosis (SPMS) frontal cortex parenchyma. Immunohistochemistry analysis of two adjacent WM fields from SPMS frontal cortex characterized, respectively, by hypertrophic astrocytes (B) and glial scar (C) reveals a noteworthy increase in P2X7R-positive astrocytes (B,C) , compared with control case (A) .

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Immunohistochemistry

    P2X7 receptor (P2X7R) mRNA and protein are augmented in secondary progressive multiple sclerosis. (A) Total RNA was extracted from three different snap-frozen blocks from MS patients (cases MS114, MS125, and MS163) and six controls (cases C12–101, C12–112, C13–010, C13–022, C14–069, and C14–053) and the expression of P2X7R mRNA was examined by RT-qPCR. Cortical protein extracts (15 μg/well) from different tissue blocks (A5C3, A5B3, A3B1, A1A2, A2A1, and A2B2) of MS cases MS114, MS125, and MS163 were analyzed by western blotting for the modulation of P2X7R, with respect to control cases (C12–101, C12–112, C13–010, C13–022, and C14–069), in both detergent-soluble (B) and -insoluble fractions (C) . GAPDH was used for protein normalization. Statistical significance was calculated by Student’s t-test, * p

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) mRNA and protein are augmented in secondary progressive multiple sclerosis. (A) Total RNA was extracted from three different snap-frozen blocks from MS patients (cases MS114, MS125, and MS163) and six controls (cases C12–101, C12–112, C13–010, C13–022, C14–069, and C14–053) and the expression of P2X7R mRNA was examined by RT-qPCR. Cortical protein extracts (15 μg/well) from different tissue blocks (A5C3, A5B3, A3B1, A1A2, A2A1, and A2B2) of MS cases MS114, MS125, and MS163 were analyzed by western blotting for the modulation of P2X7R, with respect to control cases (C12–101, C12–112, C13–010, C13–022, and C14–069), in both detergent-soluble (B) and -insoluble fractions (C) . GAPDH was used for protein normalization. Statistical significance was calculated by Student’s t-test, * p

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: In Vitro, Quantitative RT-PCR, Isolation, Flow Cytometry

    Kv7.1 and Kv7.5 expression in optimal cutting temperature compound (OCT)-embedded malignant vascular tumors. Consecutive sections of vascular tumor samples were stained by hematoxylin-eosin (HE) and immunofluorescent labeling for Kv7.1 and Kv7.5. ( Aa – Ac ) Cutaneous angiosarcoma. ( Ba – Bc ) Epithelioid haemangioendothelioma. ( Ca – Cc ) Testicular teratoma-derived angiosarcoma. ( Aa , Ba , Ca ) HE was used to identify structures. BV: healthy blood vessels; *: tumoral blood vessels. Scale bars 150 μm. Representative immunolabeling images showing merged Kv7.1 ( Ab , Bb , Cb ) and Kv7.5 ( Ac , Bc , Cc ) and tumor biopsies. Red, channels; green, α-actin (muscle); blue, DAPI nuclear staining. Scale bars 150 μm. ( D ) Tumor-to-normal (TN) ratios, in arbitrary units, were calculated from the protein expression levels obtained in immunofluorescence assays and represented in a forest plot. The analysis was performed for Kv7.1 ( top ) and Kv7.5 ( bottom ) channels, plotted in horizontal bars. The reference value (1) is indicated by a dotted line. Squares represent the mean ± SEM of the TN ratio for each patient and tumor. Gray, cutaneous angiosarcoma. White, epithelioid haemangioendothelioma. Black, testicular teratoma-derived angiosarcoma. Mean ± SEM, only represented when multiple samples and regions of interest (ROIs) were available.

    Journal: International Journal of Molecular Sciences

    Article Title: Remodeling of Kv7.1 and Kv7.5 Expression in Vascular Tumors

    doi: 10.3390/ijms21176019

    Figure Lengend Snippet: Kv7.1 and Kv7.5 expression in optimal cutting temperature compound (OCT)-embedded malignant vascular tumors. Consecutive sections of vascular tumor samples were stained by hematoxylin-eosin (HE) and immunofluorescent labeling for Kv7.1 and Kv7.5. ( Aa – Ac ) Cutaneous angiosarcoma. ( Ba – Bc ) Epithelioid haemangioendothelioma. ( Ca – Cc ) Testicular teratoma-derived angiosarcoma. ( Aa , Ba , Ca ) HE was used to identify structures. BV: healthy blood vessels; *: tumoral blood vessels. Scale bars 150 μm. Representative immunolabeling images showing merged Kv7.1 ( Ab , Bb , Cb ) and Kv7.5 ( Ac , Bc , Cc ) and tumor biopsies. Red, channels; green, α-actin (muscle); blue, DAPI nuclear staining. Scale bars 150 μm. ( D ) Tumor-to-normal (TN) ratios, in arbitrary units, were calculated from the protein expression levels obtained in immunofluorescence assays and represented in a forest plot. The analysis was performed for Kv7.1 ( top ) and Kv7.5 ( bottom ) channels, plotted in horizontal bars. The reference value (1) is indicated by a dotted line. Squares represent the mean ± SEM of the TN ratio for each patient and tumor. Gray, cutaneous angiosarcoma. White, epithelioid haemangioendothelioma. Black, testicular teratoma-derived angiosarcoma. Mean ± SEM, only represented when multiple samples and regions of interest (ROIs) were available.

    Article Snippet: After blocking for 1 h (5% nonfat powder milk, 10% goat serum, and 0.3% Triton X-100 in PBS-G), samples were incubated with anti-Kv7.1 (1:200, Alomone) or anti-Kv7.5 (1:200, Alomone) and anti-α-actin (1:500, Alomone) antibodies.

    Techniques: Expressing, Staining, Labeling, Derivative Assay, Immunolabeling, Immunofluorescence

    Voltage-dependent potassium (Kv)7.1 and Kv7.5 are differentially expressed in human blood vessels. ( A ) Membrane protein extracts were obtained from several human blood vessels (aorta and carotid arteries and inferior cava and subclavian veins) and rat tissue samples (brain, skeletal muscle and heart). ( B and C ) Fresh human samples were obtained and processed as described in the Materials and Methods. Sections were stained with anti-Kv7.1 ( B ) or anti-Kv7.5 ( C ) antibodies. Then, α-actin was used to identify muscle structures. Red, Kv7.1 ( B ) and Kv7.5 ( C ) channels; green, α-actin; merge, superimposed channel, α-actin and nuclear DAPI staining (in blue). TI: tunica intima; TM: tunica media; TE: tunica externa; e: endothelial layer; se: subendothelial layer. Scale bars: 50 μm in cava and subclavian veins; 100 μm in carotid artery; 250 μm in aorta artery.

    Journal: International Journal of Molecular Sciences

    Article Title: Remodeling of Kv7.1 and Kv7.5 Expression in Vascular Tumors

    doi: 10.3390/ijms21176019

    Figure Lengend Snippet: Voltage-dependent potassium (Kv)7.1 and Kv7.5 are differentially expressed in human blood vessels. ( A ) Membrane protein extracts were obtained from several human blood vessels (aorta and carotid arteries and inferior cava and subclavian veins) and rat tissue samples (brain, skeletal muscle and heart). ( B and C ) Fresh human samples were obtained and processed as described in the Materials and Methods. Sections were stained with anti-Kv7.1 ( B ) or anti-Kv7.5 ( C ) antibodies. Then, α-actin was used to identify muscle structures. Red, Kv7.1 ( B ) and Kv7.5 ( C ) channels; green, α-actin; merge, superimposed channel, α-actin and nuclear DAPI staining (in blue). TI: tunica intima; TM: tunica media; TE: tunica externa; e: endothelial layer; se: subendothelial layer. Scale bars: 50 μm in cava and subclavian veins; 100 μm in carotid artery; 250 μm in aorta artery.

    Article Snippet: After blocking for 1 h (5% nonfat powder milk, 10% goat serum, and 0.3% Triton X-100 in PBS-G), samples were incubated with anti-Kv7.1 (1:200, Alomone) or anti-Kv7.5 (1:200, Alomone) and anti-α-actin (1:500, Alomone) antibodies.

    Techniques: Staining

    Kv7.1 and Kv7.5 expression in paraffin-embedded vascular tumors. Consecutive sections of vascular tumor samples were stained with hematoxylin-eosin (HE) and were immunofluorescently labeled for Kv7.1 and Kv7.5. ( Aa – Ae ) Cavernous angioma. ( Ba – Be ) Capillary hemangioma. ( Ca – Ce ) Kaposi’s sarcoma. ( Da – De ) Angiosarcoma. ( Ea – Ee ) Glomus tumor. HE was used to identify structures. BV: healthy blood vessels; *: tumoral blood vessels. Scale bars: 50 μm, capillary angioma; 100 μm, cavernous angioma, Kaposi’s sarcoma and angiosarcoma; 200 μm, glomus tumor. Representative immunolabeling images showing merged Kv7.1 in healthy ( Ab , Bb , Cb , Db , Eb ) and tumor ( Ac , Bc , Cc , Dc , Ec ) biopsies. Representative immunolabeling images showing merged Kv7.5 in healthy (Ad, Bd, Cd, Dd, Ed) and tumor ( Ae , Be , Ce , De , Ee ) biopsies. Red, channels; green, α-actin (muscle); blue, DAPI nuclear staining; gray, far red detection of red blood cells showing high autofluorescence Scale bars: 100 μm in cavernous and capillary angioma; 80 μm in Kaposi’s sarcoma, angiosarcoma and glomus tumor.

    Journal: International Journal of Molecular Sciences

    Article Title: Remodeling of Kv7.1 and Kv7.5 Expression in Vascular Tumors

    doi: 10.3390/ijms21176019

    Figure Lengend Snippet: Kv7.1 and Kv7.5 expression in paraffin-embedded vascular tumors. Consecutive sections of vascular tumor samples were stained with hematoxylin-eosin (HE) and were immunofluorescently labeled for Kv7.1 and Kv7.5. ( Aa – Ae ) Cavernous angioma. ( Ba – Be ) Capillary hemangioma. ( Ca – Ce ) Kaposi’s sarcoma. ( Da – De ) Angiosarcoma. ( Ea – Ee ) Glomus tumor. HE was used to identify structures. BV: healthy blood vessels; *: tumoral blood vessels. Scale bars: 50 μm, capillary angioma; 100 μm, cavernous angioma, Kaposi’s sarcoma and angiosarcoma; 200 μm, glomus tumor. Representative immunolabeling images showing merged Kv7.1 in healthy ( Ab , Bb , Cb , Db , Eb ) and tumor ( Ac , Bc , Cc , Dc , Ec ) biopsies. Representative immunolabeling images showing merged Kv7.5 in healthy (Ad, Bd, Cd, Dd, Ed) and tumor ( Ae , Be , Ce , De , Ee ) biopsies. Red, channels; green, α-actin (muscle); blue, DAPI nuclear staining; gray, far red detection of red blood cells showing high autofluorescence Scale bars: 100 μm in cavernous and capillary angioma; 80 μm in Kaposi’s sarcoma, angiosarcoma and glomus tumor.

    Article Snippet: After blocking for 1 h (5% nonfat powder milk, 10% goat serum, and 0.3% Triton X-100 in PBS-G), samples were incubated with anti-Kv7.1 (1:200, Alomone) or anti-Kv7.5 (1:200, Alomone) and anti-α-actin (1:500, Alomone) antibodies.

    Techniques: Expressing, Staining, Labeling, Immunolabeling

    Neurite outgrowth is regulated by cAMP signaling. A. Plated neurospheres were treated with 10 μM forskolin for 24 h. The number of neurites crossing a circular mask around the neurosphere was used for quantification. B. Micrographs of control and 10 μM forskolin-treated neurospheres immunostained for beta III tubulin (green). Nuclei counterstained with DAPI (blue). C. Number of neurites crossing the circle (**p

    Journal: bioRxiv

    Article Title: Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres

    doi: 10.1101/2021.02.21.432144

    Figure Lengend Snippet: Neurite outgrowth is regulated by cAMP signaling. A. Plated neurospheres were treated with 10 μM forskolin for 24 h. The number of neurites crossing a circular mask around the neurosphere was used for quantification. B. Micrographs of control and 10 μM forskolin-treated neurospheres immunostained for beta III tubulin (green). Nuclei counterstained with DAPI (blue). C. Number of neurites crossing the circle (**p

    Article Snippet: 10-day old neurospheres were treated with 10 μM forskolin (Alomone Labs) 24 hours after plating and analyzed 24 h after treatment.

    Techniques:

    CREB phosphorylation mediates AKT2-regulated induction of PEPCK. ( A ) CREB phosphorylation induced by PTEN loss is regulated by AKT2. Liver lysates from mice with different genotypes were analysis for p-CREB. PM, Pten deleted; DM, Pten and Akt2 deleted; AM, Akt2 deleted. Two biological replicas are included for each genotype group. Protein levels of p-AKT, PTEN and p-GSK3 are analyzed and showed up-regulation of AKT and GSK-3 with PTEN loss and inhibition when AKT2 is lost. Phosphorylation of CREB is only observed when PTEN is lost. AKT2 loss together with PTEN blocked this phosphorylation in the DM livers. ( B ) Akt2 +/+ and Akt2 − / − cell were treated with forskolin (100 μM) for 24 h to induce cAMP and CREB phosphorylation or vehicle as controls. Phosphorylation of CREB is detected and showed to be lower when AKT2 is lost. When forskolin is added to induce cAMP, protein expression of p-CREB is evaluated in both Akt2 +/+ and Akt2 − / − cells even though its levels remains lower in the Akt2 − / − cells. Vinculin is probed as loading control. ( C ) Introduction of CREB-133A and wtCREB to Akt2 +/+ and Akt2 −/− hepatocytes. Expression of PEPCK is analyzed using quantitative PCR analysis. Left panel, CREB-S133A is introduced to Akt2 +/+ cells. Middle panel, wtCREB is introduced to Akt2 −/− cells; Right panel, wtCREB is introduced to Akt2 +/+ cells. n = 3. Data presented as mean ± SEM. * P ≤ 0.05. Different from vector transfected cells of the same genotype. Experiments repeated at least three times.

    Journal: The Biochemical journal

    Article Title: Regulation of basal expression of hepatic PEPCK and G6Pase by AKT2

    doi: 10.1042/BCJ20190570

    Figure Lengend Snippet: CREB phosphorylation mediates AKT2-regulated induction of PEPCK. ( A ) CREB phosphorylation induced by PTEN loss is regulated by AKT2. Liver lysates from mice with different genotypes were analysis for p-CREB. PM, Pten deleted; DM, Pten and Akt2 deleted; AM, Akt2 deleted. Two biological replicas are included for each genotype group. Protein levels of p-AKT, PTEN and p-GSK3 are analyzed and showed up-regulation of AKT and GSK-3 with PTEN loss and inhibition when AKT2 is lost. Phosphorylation of CREB is only observed when PTEN is lost. AKT2 loss together with PTEN blocked this phosphorylation in the DM livers. ( B ) Akt2 +/+ and Akt2 − / − cell were treated with forskolin (100 μM) for 24 h to induce cAMP and CREB phosphorylation or vehicle as controls. Phosphorylation of CREB is detected and showed to be lower when AKT2 is lost. When forskolin is added to induce cAMP, protein expression of p-CREB is evaluated in both Akt2 +/+ and Akt2 − / − cells even though its levels remains lower in the Akt2 − / − cells. Vinculin is probed as loading control. ( C ) Introduction of CREB-133A and wtCREB to Akt2 +/+ and Akt2 −/− hepatocytes. Expression of PEPCK is analyzed using quantitative PCR analysis. Left panel, CREB-S133A is introduced to Akt2 +/+ cells. Middle panel, wtCREB is introduced to Akt2 −/− cells; Right panel, wtCREB is introduced to Akt2 +/+ cells. n = 3. Data presented as mean ± SEM. * P ≤ 0.05. Different from vector transfected cells of the same genotype. Experiments repeated at least three times.

    Article Snippet: The medium was replaced with fresh medium with 100 μM Forskolin (Alomone Labs Cat# F-500) or vehicle and incubated for 24 h before harvesting.

    Techniques: Mouse Assay, Inhibition, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection