trichostatin a  (Alomone Labs)


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    Structured Review

    Alomone Labs trichostatin a
    DNA demethylating agents but not histone acetylation inhibition revert the IFN-γ inhibitory effect. C57BL/6 mice received 2.5 × 1011 vg/kg of AAV8-luc vector alone or in combination with 1.5 × 109 vg/kg of AAV8-IL12 vector. One group of 5 mice treated with AAV8-luc or AAV8-luc in combination with AAV8-IL12 received 5′-Azacytidine (AZA) intraperitoneally at a dose of 1 mg/kg every 24 hours starting the day of vector injection. A second group of 5 mice treated with AAV8-luc or AAV8-luc in combination with AAV8-IL12 received 2 mg/kg of <t>trichostatin</t> A (TSA) were injected IP every 48 hours starting the day of vector injection. A third group of animals received only vector injection. Luciferase expression levels were analyzed in both groups of animals at days 1, 4, and 7 using a Xenogen in vivo luminometer, experiments could not be performed at longer time points due to the toxicity of 5-AZA long term treatment. Luciferase expression was quantified and represented as photons/second. The data are shown as mean ± standard deviation (SD). The differences in luciferase expression were statistically evaluated by Student t test (**p
    Trichostatin A, 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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    Images

    1) Product Images from "IL12-Mediated Liver Inflammation Reduces the Formation of AAV Transcriptionally Active Forms but Has No Effect over Preexisting AAV Transgene Expression"

    Article Title: IL12-Mediated Liver Inflammation Reduces the Formation of AAV Transcriptionally Active Forms but Has No Effect over Preexisting AAV Transgene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067748

    DNA demethylating agents but not histone acetylation inhibition revert the IFN-γ inhibitory effect. C57BL/6 mice received 2.5 × 1011 vg/kg of AAV8-luc vector alone or in combination with 1.5 × 109 vg/kg of AAV8-IL12 vector. One group of 5 mice treated with AAV8-luc or AAV8-luc in combination with AAV8-IL12 received 5′-Azacytidine (AZA) intraperitoneally at a dose of 1 mg/kg every 24 hours starting the day of vector injection. A second group of 5 mice treated with AAV8-luc or AAV8-luc in combination with AAV8-IL12 received 2 mg/kg of trichostatin A (TSA) were injected IP every 48 hours starting the day of vector injection. A third group of animals received only vector injection. Luciferase expression levels were analyzed in both groups of animals at days 1, 4, and 7 using a Xenogen in vivo luminometer, experiments could not be performed at longer time points due to the toxicity of 5-AZA long term treatment. Luciferase expression was quantified and represented as photons/second. The data are shown as mean ± standard deviation (SD). The differences in luciferase expression were statistically evaluated by Student t test (**p
    Figure Legend Snippet: DNA demethylating agents but not histone acetylation inhibition revert the IFN-γ inhibitory effect. C57BL/6 mice received 2.5 × 1011 vg/kg of AAV8-luc vector alone or in combination with 1.5 × 109 vg/kg of AAV8-IL12 vector. One group of 5 mice treated with AAV8-luc or AAV8-luc in combination with AAV8-IL12 received 5′-Azacytidine (AZA) intraperitoneally at a dose of 1 mg/kg every 24 hours starting the day of vector injection. A second group of 5 mice treated with AAV8-luc or AAV8-luc in combination with AAV8-IL12 received 2 mg/kg of trichostatin A (TSA) were injected IP every 48 hours starting the day of vector injection. A third group of animals received only vector injection. Luciferase expression levels were analyzed in both groups of animals at days 1, 4, and 7 using a Xenogen in vivo luminometer, experiments could not be performed at longer time points due to the toxicity of 5-AZA long term treatment. Luciferase expression was quantified and represented as photons/second. The data are shown as mean ± standard deviation (SD). The differences in luciferase expression were statistically evaluated by Student t test (**p

    Techniques Used: Inhibition, Mouse Assay, Plasmid Preparation, Injection, Luciferase, Expressing, In Vivo, Standard Deviation

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    Alomone Labs anti trpa1 primary antibodies
    Axonal transport of TRPV1 and <t>TRPA1.</t> (A) TRPV1 and (C) TRPA1 immunolabeling proximal to the ligation site in saline-treated, neuritis and vinblastine-treated groups. The ligation was immediately distal to the treatment site. (B, D) Mean ratio of TRPV1 immunolabeling (ligated portion / equivalent unligated portion). P = proximal, D = distal, L = ligated portion of the nerve, UL = unligated portion of the nerve. Insert: Methods schematic showing sciatic nerve in the thigh. Note the red box represents the approximate position of the sections. D = distal, L = ligated portion of the nerve, UL = unligated portion of the nerve. Arrow head = Ligation site. * p
    Anti Trpa1 Primary Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rabbit α hcn2 antibody
    Domains mediating the interaction of VAPB with <t>HCN2.</t> A ) Schematic illustration of a HCN subunit. The CNBD and some of the truncation constructs studied are indicated. B ) All truncation constructs exhibited a positive interaction, evident from growth on -LWHA dropout medium. C ) Representative current traces and the relative currents for different C-terminal deletions expressed alone or with VAPB. D ) Representative current traces and the relative current amplitudes for the N-terminal truncated NTK HCN2 expressed alone or with VAPB. E ) Relative current amplitudes of NTK HCN2 HA Ex (extracellular HA-tag) expressed alone or with VAPB. F ) Relative surface expression of NTK HCN2 HA Ex expressed alone or with VAPB analyzed as relative light units (RLUs). G ) Schematic illustration, representative traces, and currents of a HCN2 channel chimera with the N terminus of HCN4 ( HCN4-N HCN2) expressed alone or with VAPB. H ) Relative currents of HCN2 expressed alone or coexpressed with VAPB (1.7 ± 0.1), TM VAPB (1.6 ± 0.2), the MSP domain (MSP VAPB ), the MSP with half of the CC domain (MSP-CC 0.5 VAPB ), or with the complete CC domain (MSP-CC VAPB ). I , J ) Relative current amplitudes of HCN2 HA Ex expressed alone or with TM VAPB (1.3 ± 0.1) ( I ) and the respective changes in the relative surface membrane expression analyzed as RLUs, using a single cell chemiluminescence assay (TM VAPB 1.8 ± 0.2) ( J ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. N.s., not significant. * P
    Rabbit α Hcn2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs ω conotoxin gvia
    GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM <t>ω-conotoxin-GVIA</t> (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P
    ω Conotoxin Gvia, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti kir6 2
    Confocal double immunofluorescence images of CD11b (red, A and D) and <t>Kir6.2</t> (green, B and E) in spinal cord sections from healthy control mice (Ctrl) or MOG 35-55 EAE mice . A slight intensity was found for Kir6.2 in healthy section showing low localization of the K ATP Kir6.2 subunit in CD11b-positive cells (white arrows, C). However, higher intensity of Kir6.2 subunit in CD11b reactive cells showing a strong colocalization of both (white arrows, F) was observed. Western blotting for Kir6.2 in total protein homogenates from lumbar-sacral and thoracic-cervical regions of the spinal cord from non-immunized control animals (control, G) and EAE mice (EAE, G) show an increase in Kir6.2 expression in EAE mice. This increase is statistically significant in the thoracic-cervical level of the spinal cord (H). Results are shown as mean ± SEM. ** p
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    Image Search Results


    Axonal transport of TRPV1 and TRPA1. (A) TRPV1 and (C) TRPA1 immunolabeling proximal to the ligation site in saline-treated, neuritis and vinblastine-treated groups. The ligation was immediately distal to the treatment site. (B, D) Mean ratio of TRPV1 immunolabeling (ligated portion / equivalent unligated portion). P = proximal, D = distal, L = ligated portion of the nerve, UL = unligated portion of the nerve. Insert: Methods schematic showing sciatic nerve in the thigh. Note the red box represents the approximate position of the sections. D = distal, L = ligated portion of the nerve, UL = unligated portion of the nerve. Arrow head = Ligation site. * p

    Journal: Neuroscience

    Article Title: Characterizing the mechanical properties of ectopic axonal receptive fields in inflamed nerves and following axonal transport disruption

    doi: 10.1016/j.neuroscience.2019.11.042

    Figure Lengend Snippet: Axonal transport of TRPV1 and TRPA1. (A) TRPV1 and (C) TRPA1 immunolabeling proximal to the ligation site in saline-treated, neuritis and vinblastine-treated groups. The ligation was immediately distal to the treatment site. (B, D) Mean ratio of TRPV1 immunolabeling (ligated portion / equivalent unligated portion). P = proximal, D = distal, L = ligated portion of the nerve, UL = unligated portion of the nerve. Insert: Methods schematic showing sciatic nerve in the thigh. Note the red box represents the approximate position of the sections. D = distal, L = ligated portion of the nerve, UL = unligated portion of the nerve. Arrow head = Ligation site. * p

    Article Snippet: Sections were blocked with 4% normal goat serum (Vector Labs, Burlingame, USA) in phosphate-buffered saline (PBS) (Sigma, UK) for 1 hour at room temperature and incubated overnight at 4 °C with polyclonal rabbit anti-TRPV1 (NB100–1617; Novus Biologicals, Abingdon, United Kingdom; diluted 1:500 in 4% normal goat serum in PBS) or anti-TRPA1 primary antibodies (ACC-03, Alomone Labs, Jerusalem, Israel; diluted 1:300 in 4% normal goat serum in PBS).

    Techniques: Immunolabeling, Ligation

    Domains mediating the interaction of VAPB with HCN2. A ) Schematic illustration of a HCN subunit. The CNBD and some of the truncation constructs studied are indicated. B ) All truncation constructs exhibited a positive interaction, evident from growth on -LWHA dropout medium. C ) Representative current traces and the relative currents for different C-terminal deletions expressed alone or with VAPB. D ) Representative current traces and the relative current amplitudes for the N-terminal truncated NTK HCN2 expressed alone or with VAPB. E ) Relative current amplitudes of NTK HCN2 HA Ex (extracellular HA-tag) expressed alone or with VAPB. F ) Relative surface expression of NTK HCN2 HA Ex expressed alone or with VAPB analyzed as relative light units (RLUs). G ) Schematic illustration, representative traces, and currents of a HCN2 channel chimera with the N terminus of HCN4 ( HCN4-N HCN2) expressed alone or with VAPB. H ) Relative currents of HCN2 expressed alone or coexpressed with VAPB (1.7 ± 0.1), TM VAPB (1.6 ± 0.2), the MSP domain (MSP VAPB ), the MSP with half of the CC domain (MSP-CC 0.5 VAPB ), or with the complete CC domain (MSP-CC VAPB ). I , J ) Relative current amplitudes of HCN2 HA Ex expressed alone or with TM VAPB (1.3 ± 0.1) ( I ) and the respective changes in the relative surface membrane expression analyzed as RLUs, using a single cell chemiluminescence assay (TM VAPB 1.8 ± 0.2) ( J ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. N.s., not significant. * P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: Domains mediating the interaction of VAPB with HCN2. A ) Schematic illustration of a HCN subunit. The CNBD and some of the truncation constructs studied are indicated. B ) All truncation constructs exhibited a positive interaction, evident from growth on -LWHA dropout medium. C ) Representative current traces and the relative currents for different C-terminal deletions expressed alone or with VAPB. D ) Representative current traces and the relative current amplitudes for the N-terminal truncated NTK HCN2 expressed alone or with VAPB. E ) Relative current amplitudes of NTK HCN2 HA Ex (extracellular HA-tag) expressed alone or with VAPB. F ) Relative surface expression of NTK HCN2 HA Ex expressed alone or with VAPB analyzed as relative light units (RLUs). G ) Schematic illustration, representative traces, and currents of a HCN2 channel chimera with the N terminus of HCN4 ( HCN4-N HCN2) expressed alone or with VAPB. H ) Relative currents of HCN2 expressed alone or coexpressed with VAPB (1.7 ± 0.1), TM VAPB (1.6 ± 0.2), the MSP domain (MSP VAPB ), the MSP with half of the CC domain (MSP-CC 0.5 VAPB ), or with the complete CC domain (MSP-CC VAPB ). I , J ) Relative current amplitudes of HCN2 HA Ex expressed alone or with TM VAPB (1.3 ± 0.1) ( I ) and the respective changes in the relative surface membrane expression analyzed as RLUs, using a single cell chemiluminescence assay (TM VAPB 1.8 ± 0.2) ( J ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. N.s., not significant. * P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: Construct, Expressing, Chemiluminescence Immunoassay

    VAPB determines surface expression and dendritic localization of HCN2. A ) Live cell imaging of HeLa cells transfected with an N-terminally EGFP-tagged HCN2 carrying an extracellular HA-epitope ( EGFP HCN2 HA Ex ) alone or cotransfected with VAPB or the TM segment of VAPB (TM VAPB ). B ) Chemiluminescence assays of fixed non-permeabilized HeLa cells, analyzing the surface expression as relative light units (RLUs) for EGFP HCN2 HA Ex alone and after cotransfection with VAPB (1.6 ± 0.1). Upper inset illustrates a representative control Western blot showing an unaltered HCN2 prote in expression. C ) Chemiluminescence surface expression assay as in B , but using TM VAPB (1.6 ± 0.1). D ) Immunocytochemistry of HA VAPB transfected cortical neurons. Endogenous HCN2 (green) is colocalizing (white) with HA VAPB (magenta) in the soma and dendrites. Anti–MAP2-staining illustrating an intact neuronal network and dendrites (blue). E ) Immunocytochemistry experiment as in D , but transfecting the ALS8 mutation HA VAPB P56S (magenta), leading to an aggregation of VAPB P56S in the soma of the neurons. Also, HCN2 fluorescence (green) was focused in the soma and dendritic localization was lost, despite an intact neuronal network (α-MAP2, blue). Scale bars, 20 µm ( A , D , E ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. ** P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: VAPB determines surface expression and dendritic localization of HCN2. A ) Live cell imaging of HeLa cells transfected with an N-terminally EGFP-tagged HCN2 carrying an extracellular HA-epitope ( EGFP HCN2 HA Ex ) alone or cotransfected with VAPB or the TM segment of VAPB (TM VAPB ). B ) Chemiluminescence assays of fixed non-permeabilized HeLa cells, analyzing the surface expression as relative light units (RLUs) for EGFP HCN2 HA Ex alone and after cotransfection with VAPB (1.6 ± 0.1). Upper inset illustrates a representative control Western blot showing an unaltered HCN2 prote in expression. C ) Chemiluminescence surface expression assay as in B , but using TM VAPB (1.6 ± 0.1). D ) Immunocytochemistry of HA VAPB transfected cortical neurons. Endogenous HCN2 (green) is colocalizing (white) with HA VAPB (magenta) in the soma and dendrites. Anti–MAP2-staining illustrating an intact neuronal network and dendrites (blue). E ) Immunocytochemistry experiment as in D , but transfecting the ALS8 mutation HA VAPB P56S (magenta), leading to an aggregation of VAPB P56S in the soma of the neurons. Also, HCN2 fluorescence (green) was focused in the soma and dendritic localization was lost, despite an intact neuronal network (α-MAP2, blue). Scale bars, 20 µm ( A , D , E ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. ** P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: Expressing, Live Cell Imaging, Transfection, Cotransfection, Western Blot, Immunocytochemistry, Staining, Mutagenesis, Fluorescence

    Codistribution of VAPs with HCN2 and contribution to thalamic I h . A – E ), Distribution of HCN2, VAPB, and VAPA mRNA in mouse brain and spinal cord. ISH analysis of HCN2, VAPB, and VAPA using DIG-labeled riboprobes, revealing mRNA expression of VAPB in cortical areas ( A ), hippocampus ( B ), thalamus ( C ), cerebellum ( D ) (arrows point to interneurons in the granular layer), and spinal cord ( E ). Note the overlapping distribution of VAPB with HCN2 and VAPA mRNA. Am, amygdala; CA, cornu ammonis; DG, dentate gyrus; DH, dorsal horn; gcl, granule cell layer; Hb, habenulae; ic, internal capsule; LG, lateral geniculate ncl.; m, molecular cell layer; pcl, Purkinje cell layer; RTh, reticular thalamic ncl.; Sth, subthalamic ncl.; VB, ventrobasal thalamus; Th, thalamus; VH, ventral horn. F ) Representative current traces elicited in slice patch-clamp experiments of the ventrobasal thalamus (VB) of wild-type animals (control) and VAPB −/− mice. G ) The I h current was significantly reduced in VAPB −/− mice (15.4 ± 1.1 pA/pF) compared with control animals (22.2 ± 2.3 pA/pF). H ) Average activation curves of the VB I h current for control and VAPB −/− mice. V 1/2 of activation for control (−91.6 ± 1.3 mV, n = 8) and VAPB −/− (−87.5 ± 1.2 mV, n = 7). Scale bars: 500 µm ( A–C , E ), 100 µm ( D ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. * P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: Codistribution of VAPs with HCN2 and contribution to thalamic I h . A – E ), Distribution of HCN2, VAPB, and VAPA mRNA in mouse brain and spinal cord. ISH analysis of HCN2, VAPB, and VAPA using DIG-labeled riboprobes, revealing mRNA expression of VAPB in cortical areas ( A ), hippocampus ( B ), thalamus ( C ), cerebellum ( D ) (arrows point to interneurons in the granular layer), and spinal cord ( E ). Note the overlapping distribution of VAPB with HCN2 and VAPA mRNA. Am, amygdala; CA, cornu ammonis; DG, dentate gyrus; DH, dorsal horn; gcl, granule cell layer; Hb, habenulae; ic, internal capsule; LG, lateral geniculate ncl.; m, molecular cell layer; pcl, Purkinje cell layer; RTh, reticular thalamic ncl.; Sth, subthalamic ncl.; VB, ventrobasal thalamus; Th, thalamus; VH, ventral horn. F ) Representative current traces elicited in slice patch-clamp experiments of the ventrobasal thalamus (VB) of wild-type animals (control) and VAPB −/− mice. G ) The I h current was significantly reduced in VAPB −/− mice (15.4 ± 1.1 pA/pF) compared with control animals (22.2 ± 2.3 pA/pF). H ) Average activation curves of the VB I h current for control and VAPB −/− mice. V 1/2 of activation for control (−91.6 ± 1.3 mV, n = 8) and VAPB −/− (−87.5 ± 1.2 mV, n = 7). Scale bars: 500 µm ( A–C , E ), 100 µm ( D ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. * P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: In Situ Hybridization, Labeling, Expressing, Patch Clamp, Mouse Assay, Activation Assay

    VAPB selectively increases HCN1 and HCN2 currents. A ) Y2H direct interaction assay. Transformation control (-LW), leucine, and tryptophan dropout. Interaction read-out (-LWHA), additional dropout of histidine and adenine. pAL-Alg5, positive control. pPR3-N, negative control. B ) Topology of VAPB. C ) GST VAPB pull-down of HCN2 EGFP using transfected HeLa cells. D ) GST VAPA, GST VAMP1, or GST VAMP2 pull-down of HCN2 EGFP using transfected HeLa cells. E ) GST VAPA pull-down of HCN2 and endogenous VAPB, using HCN2 EGFP transfected HeLa cells. F ) Pull-down of in vitro translated HCN2 (untagged). G ) Pull-down of HCN2 from rat brain lysates. H , I ) Representative currents ( H ) of HCN2 expressed in oocytes alone or with VAPB and the relative current amplitudes ( I ) analyzed over 3 d. J ) Relative currents of HCN1, HCN2, and HCN4 alone or coexpressed with VAPB. K , L ) Relative currents of different potassium channels ( K ) coexpressed with VAPB and of HCN2 ( L ) coexpressed with VAPA, VAPB, or VAPC. M ) Relative currents of HCN2 coexpressed with a mixture of VAPA/B (1:1). N ) Representative macropatch recordings in different configurations: on cell (o.c.), inside-out after patch excision (i.o.), and after application of 100 µM cAMP (i.o.+100 µM cAMP). O , P ) Activation curves for HCN2 alone ( n = 6) ( O ), recorded as in N , or after coexpression with VAPB ( n = 8) ( P ). Q ) V 1/2 values for HCN2 expressed alone or with VAPB in different patch modes. R ) Relative currents of HCN2 HA Ex alone or with VAPB. S ) Relative surface expression of HCN2 HA Ex expressed alone or with VAPB, analyzed as relative light units (RLUs). T ) Relative currents of HCN2 expressed alone or with VAPB or VAPB P56S . All data are presented as means ± sem . The number of cells ( n ) is indicated in the bar graphs. N.s., not significant. * P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: VAPB selectively increases HCN1 and HCN2 currents. A ) Y2H direct interaction assay. Transformation control (-LW), leucine, and tryptophan dropout. Interaction read-out (-LWHA), additional dropout of histidine and adenine. pAL-Alg5, positive control. pPR3-N, negative control. B ) Topology of VAPB. C ) GST VAPB pull-down of HCN2 EGFP using transfected HeLa cells. D ) GST VAPA, GST VAMP1, or GST VAMP2 pull-down of HCN2 EGFP using transfected HeLa cells. E ) GST VAPA pull-down of HCN2 and endogenous VAPB, using HCN2 EGFP transfected HeLa cells. F ) Pull-down of in vitro translated HCN2 (untagged). G ) Pull-down of HCN2 from rat brain lysates. H , I ) Representative currents ( H ) of HCN2 expressed in oocytes alone or with VAPB and the relative current amplitudes ( I ) analyzed over 3 d. J ) Relative currents of HCN1, HCN2, and HCN4 alone or coexpressed with VAPB. K , L ) Relative currents of different potassium channels ( K ) coexpressed with VAPB and of HCN2 ( L ) coexpressed with VAPA, VAPB, or VAPC. M ) Relative currents of HCN2 coexpressed with a mixture of VAPA/B (1:1). N ) Representative macropatch recordings in different configurations: on cell (o.c.), inside-out after patch excision (i.o.), and after application of 100 µM cAMP (i.o.+100 µM cAMP). O , P ) Activation curves for HCN2 alone ( n = 6) ( O ), recorded as in N , or after coexpression with VAPB ( n = 8) ( P ). Q ) V 1/2 values for HCN2 expressed alone or with VAPB in different patch modes. R ) Relative currents of HCN2 HA Ex alone or with VAPB. S ) Relative surface expression of HCN2 HA Ex expressed alone or with VAPB, analyzed as relative light units (RLUs). T ) Relative currents of HCN2 expressed alone or with VAPB or VAPB P56S . All data are presented as means ± sem . The number of cells ( n ) is indicated in the bar graphs. N.s., not significant. * P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: Transformation Assay, Positive Control, Negative Control, Transfection, In Vitro, Activation Assay, Expressing

    GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P

    Journal: The Journal of General Physiology

    Article Title: Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

    doi: 10.1085/jgp.201511383

    Figure Lengend Snippet: GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P

    Article Snippet: We used ghrelin esterified with n -octanoic acid (Global Peptide); a GHSR1a inverse agonist, [d -Arg1,d -Phe5,d -Trp7,9,Leu11]–substance P (SPA; Santa Cruz Biotechnology, Inc.); the inhibitor of Gs protein, cholera toxin (ChTx; Sigma Aldrich); a specific inhibitor of Gi/o protein, pertussis toxin (PTx; Sigma-Aldrich); the CaV 2.1 blocker, ω-agatoxin-IVA (Peptides International); and the CaV2.2 blocker, ω-conotoxin-GVIA (Alomone Labs).

    Techniques: Activity Assay, Mouse Assay, Activation Assay

    GHSR1a activity inhibits native Ca V 2 currents from rat hypothalamic neurons. (A) Representative and averaged I Ba from nontransfected (nt) and GFP-, GHSR1a-YFP–, and GHSR1a-A204E-YFP–transfected neurons. (B) Normalized I Ba traces before (control) and after (+ghrelin) 500-nM ghrelin application, and averaged percentage of I Ba inhibition by ghrelin in each condition. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons (left). Averaged percentage of I Ba sensitive to agaTx and conoTx from nontransfected (nt), GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from nontransfected (nt) and GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons. ANOVA with Dunnett’s post-test (A–D). *, P

    Journal: The Journal of General Physiology

    Article Title: Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

    doi: 10.1085/jgp.201511383

    Figure Lengend Snippet: GHSR1a activity inhibits native Ca V 2 currents from rat hypothalamic neurons. (A) Representative and averaged I Ba from nontransfected (nt) and GFP-, GHSR1a-YFP–, and GHSR1a-A204E-YFP–transfected neurons. (B) Normalized I Ba traces before (control) and after (+ghrelin) 500-nM ghrelin application, and averaged percentage of I Ba inhibition by ghrelin in each condition. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons (left). Averaged percentage of I Ba sensitive to agaTx and conoTx from nontransfected (nt), GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from nontransfected (nt) and GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons. ANOVA with Dunnett’s post-test (A–D). *, P

    Article Snippet: We used ghrelin esterified with n -octanoic acid (Global Peptide); a GHSR1a inverse agonist, [d -Arg1,d -Phe5,d -Trp7,9,Leu11]–substance P (SPA; Santa Cruz Biotechnology, Inc.); the inhibitor of Gs protein, cholera toxin (ChTx; Sigma Aldrich); a specific inhibitor of Gi/o protein, pertussis toxin (PTx; Sigma-Aldrich); the CaV 2.1 blocker, ω-agatoxin-IVA (Peptides International); and the CaV2.2 blocker, ω-conotoxin-GVIA (Alomone Labs).

    Techniques: Activity Assay, Transfection, Inhibition

    Confocal double immunofluorescence images of CD11b (red, A and D) and Kir6.2 (green, B and E) in spinal cord sections from healthy control mice (Ctrl) or MOG 35-55 EAE mice . A slight intensity was found for Kir6.2 in healthy section showing low localization of the K ATP Kir6.2 subunit in CD11b-positive cells (white arrows, C). However, higher intensity of Kir6.2 subunit in CD11b reactive cells showing a strong colocalization of both (white arrows, F) was observed. Western blotting for Kir6.2 in total protein homogenates from lumbar-sacral and thoracic-cervical regions of the spinal cord from non-immunized control animals (control, G) and EAE mice (EAE, G) show an increase in Kir6.2 expression in EAE mice. This increase is statistically significant in the thoracic-cervical level of the spinal cord (H). Results are shown as mean ± SEM. ** p

    Journal: Journal of Neuroinflammation

    Article Title: Oral administration of the KATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

    doi: 10.1186/1742-2094-8-149

    Figure Lengend Snippet: Confocal double immunofluorescence images of CD11b (red, A and D) and Kir6.2 (green, B and E) in spinal cord sections from healthy control mice (Ctrl) or MOG 35-55 EAE mice . A slight intensity was found for Kir6.2 in healthy section showing low localization of the K ATP Kir6.2 subunit in CD11b-positive cells (white arrows, C). However, higher intensity of Kir6.2 subunit in CD11b reactive cells showing a strong colocalization of both (white arrows, F) was observed. Western blotting for Kir6.2 in total protein homogenates from lumbar-sacral and thoracic-cervical regions of the spinal cord from non-immunized control animals (control, G) and EAE mice (EAE, G) show an increase in Kir6.2 expression in EAE mice. This increase is statistically significant in the thoracic-cervical level of the spinal cord (H). Results are shown as mean ± SEM. ** p

    Article Snippet: Cells were then incubated with primary antibodies anti-Kir6.1 and anti-Kir6.2 (1:300 dilution, Alomone, Jerusalem, Israel), anti-CD11b (1:500 dilution, Serotec, Oxford, England, UK) at 4°C overnight, followed by secondary antibodies Alexa®488 and 596 (1:500, Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h in blocking solution.

    Techniques: Immunofluorescence, Mouse Assay, Western Blot, Expressing

    Western blotting show expression of both Kir6.1 and Kir6.2 K ATP channel pore-forming subunits in unstimulated and LPS/IFNγ-stimulated BV-2 cells (A, left) and microglial primary cultures (A, Right). Staining for the microglial cell membrane marker CD11b (B and E) and the K ATP channel subunits Kir 6.1 (C) or Kir 6.2 (F) showed colocalization in BV-2 microglia, indicating the expression of the K ATP channel at the cytoplasmic membrane (D and G, white arrows) . Control: unstimulated cells; L+I: cells stimulated with LPS and IFNγ. Scale bar = 30 μm.

    Journal: Journal of Neuroinflammation

    Article Title: Oral administration of the KATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

    doi: 10.1186/1742-2094-8-149

    Figure Lengend Snippet: Western blotting show expression of both Kir6.1 and Kir6.2 K ATP channel pore-forming subunits in unstimulated and LPS/IFNγ-stimulated BV-2 cells (A, left) and microglial primary cultures (A, Right). Staining for the microglial cell membrane marker CD11b (B and E) and the K ATP channel subunits Kir 6.1 (C) or Kir 6.2 (F) showed colocalization in BV-2 microglia, indicating the expression of the K ATP channel at the cytoplasmic membrane (D and G, white arrows) . Control: unstimulated cells; L+I: cells stimulated with LPS and IFNγ. Scale bar = 30 μm.

    Article Snippet: Cells were then incubated with primary antibodies anti-Kir6.1 and anti-Kir6.2 (1:300 dilution, Alomone, Jerusalem, Israel), anti-CD11b (1:500 dilution, Serotec, Oxford, England, UK) at 4°C overnight, followed by secondary antibodies Alexa®488 and 596 (1:500, Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h in blocking solution.

    Techniques: Western Blot, Expressing, Staining, Marker