guinea pig anti bdnf antibody  (Alomone Labs)


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    Name:
    Guinea pig Anti BDNF Antibody
    Description:
    Guinea pig Anti BDNF Antibody AGP 021 is a highly specific antibody directed against an epitope of human brain derived neurotrophic factor The antibody can be used in western blot and immunohistochemistry applications It has been designed to recognize BDNF from rat human and mouse samples The antibody is specific for BDNF it does not crossreact with NGF NT 3 or NT 4 The antigen used to immunize guinea pigs is the same as Anti BDNF Antibody ANT 010 raised in rabbit Our line of guinea pig antibodies enables more flexibility with our products such as multiplex staining studies immunoprecipitation etc
    Catalog Number:
    AGP-021
    Price:
    397.0
    Category:
    Primary Antibody
    Applications:
    Immunofluorescence, Immunohistochemistry, Western Blot
    Purity:
    Affinity purified on immobilized antigen.
    Immunogen:
    Synthetic peptide
    Size:
    25 mcl
    Antibody Type:
    Polyclonal Primary Antibodies
    Format:
    Lyophilized Powder
    Host:
    Guinea pig
    Isotype:
    Guinea pig total IgG
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    Structured Review

    Alomone Labs guinea pig anti bdnf antibody
    Guinea pig Anti BDNF Antibody
    Guinea pig Anti BDNF Antibody AGP 021 is a highly specific antibody directed against an epitope of human brain derived neurotrophic factor The antibody can be used in western blot and immunohistochemistry applications It has been designed to recognize BDNF from rat human and mouse samples The antibody is specific for BDNF it does not crossreact with NGF NT 3 or NT 4 The antigen used to immunize guinea pigs is the same as Anti BDNF Antibody ANT 010 raised in rabbit Our line of guinea pig antibodies enables more flexibility with our products such as multiplex staining studies immunoprecipitation etc
    https://www.bioz.com/result/guinea pig anti bdnf antibody/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    guinea pig anti bdnf antibody - by Bioz Stars, 2021-09
    92/100 stars

    Images

    1) Product Images from "Telomerase increasing compound protects hippocampal neurons from amyloid beta toxicity by enhancing the expression of neurotrophins and plasticity related genes"

    Article Title: Telomerase increasing compound protects hippocampal neurons from amyloid beta toxicity by enhancing the expression of neurotrophins and plasticity related genes

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-54741-7

    AGS increases BDNF but not NGF gene expression in both Aβ treated and untreated cultures. Cultures were treated for 36 hr. with AGS-499 (200 nM) alone, Aβ (5 µM) alone, and Aβ (5 µM) in combination with AGS-499 (200 nM) or with its vehicle DMSO. AGS or DMSO treatments were renewed after 24 hr. Expression levels were quantified using RT-PCR and normalized to the untreated control (Control). ( a ) Relative BDNF gene expression in cultures (Mean ± SEM, n = 5 independent experiments), ANOVA test ***p
    Figure Legend Snippet: AGS increases BDNF but not NGF gene expression in both Aβ treated and untreated cultures. Cultures were treated for 36 hr. with AGS-499 (200 nM) alone, Aβ (5 µM) alone, and Aβ (5 µM) in combination with AGS-499 (200 nM) or with its vehicle DMSO. AGS or DMSO treatments were renewed after 24 hr. Expression levels were quantified using RT-PCR and normalized to the untreated control (Control). ( a ) Relative BDNF gene expression in cultures (Mean ± SEM, n = 5 independent experiments), ANOVA test ***p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Related Articles

    Incubation:

    Article Title: Daytime Light Intensity Modulates Spatial Learning and Hippocampal Plasticity in Female Nile Grass Rats (Arvicanthis niloticus)
    Article Snippet: .. Membranes were incubated in Odyssey Blocking Buffer TBS (OBB-TBS) on shaker for 1 hour at room temperature, followed by incubation in guinea pig anti-BNDF primary antibody (1:1000; AGP-021; Alomone Labs, Jerusalem, Israel) at 4 °C for 5 days. ..

    Blocking Assay:

    Article Title: Daytime Light Intensity Modulates Spatial Learning and Hippocampal Plasticity in Female Nile Grass Rats (Arvicanthis niloticus)
    Article Snippet: .. Membranes were incubated in Odyssey Blocking Buffer TBS (OBB-TBS) on shaker for 1 hour at room temperature, followed by incubation in guinea pig anti-BNDF primary antibody (1:1000; AGP-021; Alomone Labs, Jerusalem, Israel) at 4 °C for 5 days. ..

    Derivative Assay:

    Article Title: Telomerase increasing compound protects hippocampal neurons from amyloid beta toxicity by enhancing the expression of neurotrophins and plasticity related genes
    Article Snippet: .. The brain slices (5 μΜ) derived from vehicle (DMSO) and AGS-499 treated mice were subjected to immunofluorescence analysis using as first antibody: Guinea pig anti BDNF-antibody (1:100, # AGP-021, alomono labs, Israel) or anti-beta catenin antibody (1:100, β-Catenin (6B3) Rabbit mAb #9582, Cell Signaling technology, Danvers, MA, USA). ..

    Mouse Assay:

    Article Title: Telomerase increasing compound protects hippocampal neurons from amyloid beta toxicity by enhancing the expression of neurotrophins and plasticity related genes
    Article Snippet: .. The brain slices (5 μΜ) derived from vehicle (DMSO) and AGS-499 treated mice were subjected to immunofluorescence analysis using as first antibody: Guinea pig anti BDNF-antibody (1:100, # AGP-021, alomono labs, Israel) or anti-beta catenin antibody (1:100, β-Catenin (6B3) Rabbit mAb #9582, Cell Signaling technology, Danvers, MA, USA). ..

    Immunofluorescence:

    Article Title: Telomerase increasing compound protects hippocampal neurons from amyloid beta toxicity by enhancing the expression of neurotrophins and plasticity related genes
    Article Snippet: .. The brain slices (5 μΜ) derived from vehicle (DMSO) and AGS-499 treated mice were subjected to immunofluorescence analysis using as first antibody: Guinea pig anti BDNF-antibody (1:100, # AGP-021, alomono labs, Israel) or anti-beta catenin antibody (1:100, β-Catenin (6B3) Rabbit mAb #9582, Cell Signaling technology, Danvers, MA, USA). ..

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    Alomone Labs guinea pig anti bdnf antibody
    <t>AGS</t> increases <t>BDNF</t> but not NGF gene expression in both Aβ treated and untreated cultures. Cultures were treated for 36 hr. with AGS-499 (200 nM) alone, Aβ (5 µM) alone, and Aβ (5 µM) in combination with AGS-499 (200 nM) or with its vehicle DMSO. AGS or DMSO treatments were renewed after 24 hr. Expression levels were quantified using RT-PCR and normalized to the untreated control (Control). ( a ) Relative BDNF gene expression in cultures (Mean ± SEM, n = 5 independent experiments), ANOVA test ***p
    Guinea Pig Anti Bdnf Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/guinea pig anti bdnf antibody/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    guinea pig anti bdnf antibody - by Bioz Stars, 2021-09
    92/100 stars
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    90
    Alomone Labs guinea pig anti hcn1
    Analyses of expression of the Cav2.3 protein in neuronal and nonneuronal cells or in cell membrane of the cells in dorsal root ganglia in vivo. Representative images to demonstrate expression of Cav2.3 in mouse DRG and colabeling with PGP9.5-positive neuronal cells (A), GFAP-positive satellite cells (B) and with <t>HCN1</t> (hyperpolarization-activated cyclic nucleotide-gated) channel in the cell membrane (C). Observed colocalization is highlighted with white arrows. Quantification of coexpression of each neuronal subtype with the Cav2.3 expressing neurons is shown in panel D. Scale bars represent 50 µm in all panels. Tissue samples from 3 independent mice were analyzed.
    Guinea Pig Anti Hcn1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/guinea pig anti hcn1/product/Alomone Labs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    91
    Alomone Labs kv1 3
    <t>Kv1.3</t> channel activity controls tight junction protein expression on bEnd.3. ( a ) Immunofluorescence analysis of Kv1.3 (in red, Hoechst in blue) on endothelial bEnd.3 cells. On the right, cells stained only with secondary Ab as control. ( b ) Astrocytes were co-cultured with bEnd.3 in the absence (−GL261) or presence (+GL261) of GL261 cells (as depicted in the inset), and treated or not with PAP-1 (50 nM). Trans-endothelial electric resistance (TEER, in Ωcm2) was measured at the indicated time points. ( c ) bEnd.3 in the absence (−GL261) or presence (+GL261) of GL261 cells (as depicted in the inset), and treated or not with PAP-1 (50 nM) were assayed for TEER (in Ωcm 2 ) at the indicated time points. ( d ) RT-PCR gene expression of claudin-5, occludin and zo-1 in untreated (C) or PAP-1 (50 nM) treated bEnd.3 co-cultured or not with astrocytes. Data are expressed as fold increase in co-cultures vs bEnd.3 alone (no astrocytes) and are the mean ± s.e.m., n = 4, *p = 0.001, Dunn’s method One Way ANOVA.
    Kv1 3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kv1 3/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    94
    Alomone Labs cav1 2
    <t>Kv1.3</t> channel activity controls tight junction protein expression on bEnd.3. ( a ) Immunofluorescence analysis of Kv1.3 (in red, Hoechst in blue) on endothelial bEnd.3 cells. On the right, cells stained only with secondary Ab as control. ( b ) Astrocytes were co-cultured with bEnd.3 in the absence (−GL261) or presence (+GL261) of GL261 cells (as depicted in the inset), and treated or not with PAP-1 (50 nM). Trans-endothelial electric resistance (TEER, in Ωcm2) was measured at the indicated time points. ( c ) bEnd.3 in the absence (−GL261) or presence (+GL261) of GL261 cells (as depicted in the inset), and treated or not with PAP-1 (50 nM) were assayed for TEER (in Ωcm 2 ) at the indicated time points. ( d ) RT-PCR gene expression of claudin-5, occludin and zo-1 in untreated (C) or PAP-1 (50 nM) treated bEnd.3 co-cultured or not with astrocytes. Data are expressed as fold increase in co-cultures vs bEnd.3 alone (no astrocytes) and are the mean ± s.e.m., n = 4, *p = 0.001, Dunn’s method One Way ANOVA.
    Cav1 2, 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
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    Average 94 stars, based on 1 article reviews
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    Image Search Results


    AGS increases BDNF but not NGF gene expression in both Aβ treated and untreated cultures. Cultures were treated for 36 hr. with AGS-499 (200 nM) alone, Aβ (5 µM) alone, and Aβ (5 µM) in combination with AGS-499 (200 nM) or with its vehicle DMSO. AGS or DMSO treatments were renewed after 24 hr. Expression levels were quantified using RT-PCR and normalized to the untreated control (Control). ( a ) Relative BDNF gene expression in cultures (Mean ± SEM, n = 5 independent experiments), ANOVA test ***p

    Journal: Scientific Reports

    Article Title: Telomerase increasing compound protects hippocampal neurons from amyloid beta toxicity by enhancing the expression of neurotrophins and plasticity related genes

    doi: 10.1038/s41598-019-54741-7

    Figure Lengend Snippet: AGS increases BDNF but not NGF gene expression in both Aβ treated and untreated cultures. Cultures were treated for 36 hr. with AGS-499 (200 nM) alone, Aβ (5 µM) alone, and Aβ (5 µM) in combination with AGS-499 (200 nM) or with its vehicle DMSO. AGS or DMSO treatments were renewed after 24 hr. Expression levels were quantified using RT-PCR and normalized to the untreated control (Control). ( a ) Relative BDNF gene expression in cultures (Mean ± SEM, n = 5 independent experiments), ANOVA test ***p

    Article Snippet: The brain slices (5 μΜ) derived from vehicle (DMSO) and AGS-499 treated mice were subjected to immunofluorescence analysis using as first antibody: Guinea pig anti BDNF-antibody (1:100, # AGP-021, alomono labs, Israel) or anti-beta catenin antibody (1:100, β-Catenin (6B3) Rabbit mAb #9582, Cell Signaling technology, Danvers, MA, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Analyses of expression of the Cav2.3 protein in neuronal and nonneuronal cells or in cell membrane of the cells in dorsal root ganglia in vivo. Representative images to demonstrate expression of Cav2.3 in mouse DRG and colabeling with PGP9.5-positive neuronal cells (A), GFAP-positive satellite cells (B) and with HCN1 (hyperpolarization-activated cyclic nucleotide-gated) channel in the cell membrane (C). Observed colocalization is highlighted with white arrows. Quantification of coexpression of each neuronal subtype with the Cav2.3 expressing neurons is shown in panel D. Scale bars represent 50 µm in all panels. Tissue samples from 3 independent mice were analyzed.

    Journal: Pain

    Article Title: miR-34c-5p functions as pronociceptive microRNA in cancer pain by targeting Cav2.3 containing calcium channels

    doi: 10.1097/j.pain.0000000000000971

    Figure Lengend Snippet: Analyses of expression of the Cav2.3 protein in neuronal and nonneuronal cells or in cell membrane of the cells in dorsal root ganglia in vivo. Representative images to demonstrate expression of Cav2.3 in mouse DRG and colabeling with PGP9.5-positive neuronal cells (A), GFAP-positive satellite cells (B) and with HCN1 (hyperpolarization-activated cyclic nucleotide-gated) channel in the cell membrane (C). Observed colocalization is highlighted with white arrows. Quantification of coexpression of each neuronal subtype with the Cav2.3 expressing neurons is shown in panel D. Scale bars represent 50 µm in all panels. Tissue samples from 3 independent mice were analyzed.

    Article Snippet: Primary antibodies used for IF are Guinea pig anti-PGP9.5 (1:100 dilution, 14104, Neuromics, Edina, MN), Guinea pig anti-HCN1 (1:100, Alomone Labs, AGP203) rabbit anti-Cav2.3 antibody (1:80, Alomone Labs, ACC-006), Biotinylated-Isolectin B4 (1:100; B-1205, Vector, Burlingame, CA), Guinea pig Substance P (1:150; Neuromics GP14103), Anti-GFAP (1:500; NeuroMab clone N206A/8, UC Davis, Davis, CA) and Chicken anti-NF200 (1:500; Neuromics CH23015).

    Techniques: Expressing, In Vivo, Mouse Assay

    Kv1.3 channel activity controls tight junction protein expression on bEnd.3. ( a ) Immunofluorescence analysis of Kv1.3 (in red, Hoechst in blue) on endothelial bEnd.3 cells. On the right, cells stained only with secondary Ab as control. ( b ) Astrocytes were co-cultured with bEnd.3 in the absence (−GL261) or presence (+GL261) of GL261 cells (as depicted in the inset), and treated or not with PAP-1 (50 nM). Trans-endothelial electric resistance (TEER, in Ωcm2) was measured at the indicated time points. ( c ) bEnd.3 in the absence (−GL261) or presence (+GL261) of GL261 cells (as depicted in the inset), and treated or not with PAP-1 (50 nM) were assayed for TEER (in Ωcm 2 ) at the indicated time points. ( d ) RT-PCR gene expression of claudin-5, occludin and zo-1 in untreated (C) or PAP-1 (50 nM) treated bEnd.3 co-cultured or not with astrocytes. Data are expressed as fold increase in co-cultures vs bEnd.3 alone (no astrocytes) and are the mean ± s.e.m., n = 4, *p = 0.001, Dunn’s method One Way ANOVA.

    Journal: Scientific Reports

    Article Title: Kv1.3 activity perturbs the homeostatic properties of astrocytes in glioma

    doi: 10.1038/s41598-018-25940-5

    Figure Lengend Snippet: Kv1.3 channel activity controls tight junction protein expression on bEnd.3. ( a ) Immunofluorescence analysis of Kv1.3 (in red, Hoechst in blue) on endothelial bEnd.3 cells. On the right, cells stained only with secondary Ab as control. ( b ) Astrocytes were co-cultured with bEnd.3 in the absence (−GL261) or presence (+GL261) of GL261 cells (as depicted in the inset), and treated or not with PAP-1 (50 nM). Trans-endothelial electric resistance (TEER, in Ωcm2) was measured at the indicated time points. ( c ) bEnd.3 in the absence (−GL261) or presence (+GL261) of GL261 cells (as depicted in the inset), and treated or not with PAP-1 (50 nM) were assayed for TEER (in Ωcm 2 ) at the indicated time points. ( d ) RT-PCR gene expression of claudin-5, occludin and zo-1 in untreated (C) or PAP-1 (50 nM) treated bEnd.3 co-cultured or not with astrocytes. Data are expressed as fold increase in co-cultures vs bEnd.3 alone (no astrocytes) and are the mean ± s.e.m., n = 4, *p = 0.001, Dunn’s method One Way ANOVA.

    Article Snippet: Immunofluorescence Coronal brain sections (20 μm) were washed in PBS, blocked (3% goat serum in 0.3% Triton X-100) for 1 h at RT and incubated overnight at 4 °C with specific antibodies, GFAP (1:750 –Novus Biologicals, NB300-141), Iba1 (1:750 - Wako, 019-19741), GLT1 (1:1000 – AbCam, ab41621), Kv1.3 (1:100 – Alomone Lab, AGP-005).

    Techniques: Activity Assay, Expressing, Immunofluorescence, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    Kv1.3 activity modulates microglia functions. ( a – b ) Non-conditioned medium (NCM)- and glioma conditioned medium (GCM)-treated microglia, in the absence (C) or presence of PAP-1 (50 nM) assayed for phagocytosis ( a ) and migration ( b ). Data are expressed as the % of phagocytosing ( a ) and migrated ( b ) cells ± s.e.m. *p = 0.001vs NCM; n = 4, Kruskal-Wallis One Way ANOVA on Ranks. ( c ) Coronal brain sections of GL261-bearing mice treated with PAP-1 (40 mg/kg/die) or vehicle were stained for Iba1 (red) and Hoechst (blue), scale bar 20 µm. On the right, % of Iba1 + cell area normalized for tumor area, *p = 0.002, unpaired t -test, n = 6. ( d , e ) RT-PCR for pro- ( cd86, tnfα, il1α, il15 ) and anti- ( arg1, ym1, cd163, cd206 ) inflammatory genes expressed by CD11b + cells extracted from ipsilateral hemisphere of brains of GL261-bearing mice treated with vehicle (C) or PAP-1 (40 mg/kg/die). Data are expressed as fold change of PAP-1-treated vs vehicle-treated samples (C, dashed lines) and are the mean ± s.e.m., *p

    Journal: Scientific Reports

    Article Title: Kv1.3 activity perturbs the homeostatic properties of astrocytes in glioma

    doi: 10.1038/s41598-018-25940-5

    Figure Lengend Snippet: Kv1.3 activity modulates microglia functions. ( a – b ) Non-conditioned medium (NCM)- and glioma conditioned medium (GCM)-treated microglia, in the absence (C) or presence of PAP-1 (50 nM) assayed for phagocytosis ( a ) and migration ( b ). Data are expressed as the % of phagocytosing ( a ) and migrated ( b ) cells ± s.e.m. *p = 0.001vs NCM; n = 4, Kruskal-Wallis One Way ANOVA on Ranks. ( c ) Coronal brain sections of GL261-bearing mice treated with PAP-1 (40 mg/kg/die) or vehicle were stained for Iba1 (red) and Hoechst (blue), scale bar 20 µm. On the right, % of Iba1 + cell area normalized for tumor area, *p = 0.002, unpaired t -test, n = 6. ( d , e ) RT-PCR for pro- ( cd86, tnfα, il1α, il15 ) and anti- ( arg1, ym1, cd163, cd206 ) inflammatory genes expressed by CD11b + cells extracted from ipsilateral hemisphere of brains of GL261-bearing mice treated with vehicle (C) or PAP-1 (40 mg/kg/die). Data are expressed as fold change of PAP-1-treated vs vehicle-treated samples (C, dashed lines) and are the mean ± s.e.m., *p

    Article Snippet: Immunofluorescence Coronal brain sections (20 μm) were washed in PBS, blocked (3% goat serum in 0.3% Triton X-100) for 1 h at RT and incubated overnight at 4 °C with specific antibodies, GFAP (1:750 –Novus Biologicals, NB300-141), Iba1 (1:750 - Wako, 019-19741), GLT1 (1:1000 – AbCam, ab41621), Kv1.3 (1:100 – Alomone Lab, AGP-005).

    Techniques: Activity Assay, Migration, Mouse Assay, Staining, Reverse Transcription Polymerase Chain Reaction

    The inhibition of Kv1.3 channels induces neuroprotection against the toxic effects of glioma. ( a ) Cortical neurons (CN) co-cultured with GL261 cells (grey bars) or alone (black bars) were treated with PAP-1 (50 nM, 18 h) or vehicle (C) and analyzed for neuronal viability. Results are expressed as number of viable cells/field. *p = 0.001 vs C; n = 4, unpaired t -test. ( b ) Hippocampal neurons (HN) pre-treated or not with empty or clodronate-filled liposomes for 24 h, co-cultured as in ( a ) for a further 18 h in presence of PAP-1 (50 nM) or vehicle (C), were analyzed for neuronal viability. Results are expressed as number of viable cells/field. **p = 0.001 and *p

    Journal: Scientific Reports

    Article Title: Kv1.3 activity perturbs the homeostatic properties of astrocytes in glioma

    doi: 10.1038/s41598-018-25940-5

    Figure Lengend Snippet: The inhibition of Kv1.3 channels induces neuroprotection against the toxic effects of glioma. ( a ) Cortical neurons (CN) co-cultured with GL261 cells (grey bars) or alone (black bars) were treated with PAP-1 (50 nM, 18 h) or vehicle (C) and analyzed for neuronal viability. Results are expressed as number of viable cells/field. *p = 0.001 vs C; n = 4, unpaired t -test. ( b ) Hippocampal neurons (HN) pre-treated or not with empty or clodronate-filled liposomes for 24 h, co-cultured as in ( a ) for a further 18 h in presence of PAP-1 (50 nM) or vehicle (C), were analyzed for neuronal viability. Results are expressed as number of viable cells/field. **p = 0.001 and *p

    Article Snippet: Immunofluorescence Coronal brain sections (20 μm) were washed in PBS, blocked (3% goat serum in 0.3% Triton X-100) for 1 h at RT and incubated overnight at 4 °C with specific antibodies, GFAP (1:750 –Novus Biologicals, NB300-141), Iba1 (1:750 - Wako, 019-19741), GLT1 (1:1000 – AbCam, ab41621), Kv1.3 (1:100 – Alomone Lab, AGP-005).

    Techniques: Inhibition, Cell Culture

    Kv1.3 is expressed by glioma cells and modulates their migration. ( a ) Typical current traces in response to repeated voltage ramps from −120 to +50 mV (holding potential −70 mV) in Ctrl and PAP-1 (100 nM) treated GL261 cells. ( b ) Bar graph representing PAP-1 sensitive current amplitude in GL261 cells, n = 14, *p = 0.01, t -test. ( c ) Migration assay on untreated (Ctrl) and PAP-1 (50 nM, 4 h) treated GL261, GL-15 and GBM18 cells; data are the mean ± s.e.m., n = 4, *p = 0.001, # p = 0.05, @ p = 0.001, unpaired t -test.

    Journal: Scientific Reports

    Article Title: Kv1.3 activity perturbs the homeostatic properties of astrocytes in glioma

    doi: 10.1038/s41598-018-25940-5

    Figure Lengend Snippet: Kv1.3 is expressed by glioma cells and modulates their migration. ( a ) Typical current traces in response to repeated voltage ramps from −120 to +50 mV (holding potential −70 mV) in Ctrl and PAP-1 (100 nM) treated GL261 cells. ( b ) Bar graph representing PAP-1 sensitive current amplitude in GL261 cells, n = 14, *p = 0.01, t -test. ( c ) Migration assay on untreated (Ctrl) and PAP-1 (50 nM, 4 h) treated GL261, GL-15 and GBM18 cells; data are the mean ± s.e.m., n = 4, *p = 0.001, # p = 0.05, @ p = 0.001, unpaired t -test.

    Article Snippet: Immunofluorescence Coronal brain sections (20 μm) were washed in PBS, blocked (3% goat serum in 0.3% Triton X-100) for 1 h at RT and incubated overnight at 4 °C with specific antibodies, GFAP (1:750 –Novus Biologicals, NB300-141), Iba1 (1:750 - Wako, 019-19741), GLT1 (1:1000 – AbCam, ab41621), Kv1.3 (1:100 – Alomone Lab, AGP-005).

    Techniques: Migration

    Kv1.3 activity modulates glutamate buffering on astrocytes. ( a ) Time course of fluorescence ratio (ΔF/F0) changes induced by a puff of glutamate (1 mM for 0.5 sec, Glut puff ) onto astrocytic cultures loaded with BCECF-AM (10 μM, 45 min) and pre-treated with vehicle (n = 71) or PAP-1 (100 nM, n = 69). At peak ΔF/F0 in PAP1 = −0.08 ± 0.008 vs −0.046 ± 0.014 in Ctrl p = 0.0009, unpaired Student’s t -test). (b) Astrocytes were treated with PAP-1 (50 nM, grey circles) or not (black circles) with or without DHK (500 μM, triangles) for different times (from 2 to 45 min) and analyzed for intracellular D-[ 3 H]Asp, as described in the Methods section. Results are expressed as pCi/μg proteins and are the mean ± s.e.m. of at least 5 triplicate experiments. *p = 0.001 vs C of the correspondent time point, Holm-Sidak method One Way ANOVA. ( c ) Confocal images of astrocytes, untreated (C) or treated with PAP-1 (50 nM, 25 min), stained for plasma membrane GLT-1 (red, Hoechst in blue), scale bar 10 μm. On the right, data represent the mean fluorescence intensity of red signals per field ± s.e.m. n = 4, *p = 0.042 vs C, unpaired t -test. ( d ) Astrocytes untreated (−) or treated (+) with PAP-1 (50 nM, 25 min) were immunoprecipitated for Sumo-1 or control IgG and immmuno-blotted for GLT-1; total lysate (input) is shown. On the right, data represent the mean ± s.e.m. of optical density of sumoylated GLT-1 expressed as % of the input, *p = 0.028 vs C, unpaired t -test. ( e ) Representative time course of spontaneous Ca 2+ oscillation (F/F0) in cultured astrocytes loaded with Fluo4-AM in CTRL (left panel) and after PAP-1 application (right panel). Each trace in the panel represent a single ROI in the field. ( f ) Quantification of Ca 2+ transients before and after PAP-1 treatment. ( g ) Average ΔF/F0 of Ca 2+ transient in CTRL condition and after PAP-1 application. ( h ) Coronal brain sections of GL261-bearing mice treated with vehicle or PAP-1 (40 mg/kg/die) were stained for GFAP (green; Hoechst, in blue) and visualized at the border of the tumor (white dashed line), scale bar 20 μm. Right, data represent the mean area (in pixels) covered by GFAP + cells present at a distance up to 100 μm from the tumor border (mean ± s.e.m. n = 6, *p = 0.015, unpaired t -test).

    Journal: Scientific Reports

    Article Title: Kv1.3 activity perturbs the homeostatic properties of astrocytes in glioma

    doi: 10.1038/s41598-018-25940-5

    Figure Lengend Snippet: Kv1.3 activity modulates glutamate buffering on astrocytes. ( a ) Time course of fluorescence ratio (ΔF/F0) changes induced by a puff of glutamate (1 mM for 0.5 sec, Glut puff ) onto astrocytic cultures loaded with BCECF-AM (10 μM, 45 min) and pre-treated with vehicle (n = 71) or PAP-1 (100 nM, n = 69). At peak ΔF/F0 in PAP1 = −0.08 ± 0.008 vs −0.046 ± 0.014 in Ctrl p = 0.0009, unpaired Student’s t -test). (b) Astrocytes were treated with PAP-1 (50 nM, grey circles) or not (black circles) with or without DHK (500 μM, triangles) for different times (from 2 to 45 min) and analyzed for intracellular D-[ 3 H]Asp, as described in the Methods section. Results are expressed as pCi/μg proteins and are the mean ± s.e.m. of at least 5 triplicate experiments. *p = 0.001 vs C of the correspondent time point, Holm-Sidak method One Way ANOVA. ( c ) Confocal images of astrocytes, untreated (C) or treated with PAP-1 (50 nM, 25 min), stained for plasma membrane GLT-1 (red, Hoechst in blue), scale bar 10 μm. On the right, data represent the mean fluorescence intensity of red signals per field ± s.e.m. n = 4, *p = 0.042 vs C, unpaired t -test. ( d ) Astrocytes untreated (−) or treated (+) with PAP-1 (50 nM, 25 min) were immunoprecipitated for Sumo-1 or control IgG and immmuno-blotted for GLT-1; total lysate (input) is shown. On the right, data represent the mean ± s.e.m. of optical density of sumoylated GLT-1 expressed as % of the input, *p = 0.028 vs C, unpaired t -test. ( e ) Representative time course of spontaneous Ca 2+ oscillation (F/F0) in cultured astrocytes loaded with Fluo4-AM in CTRL (left panel) and after PAP-1 application (right panel). Each trace in the panel represent a single ROI in the field. ( f ) Quantification of Ca 2+ transients before and after PAP-1 treatment. ( g ) Average ΔF/F0 of Ca 2+ transient in CTRL condition and after PAP-1 application. ( h ) Coronal brain sections of GL261-bearing mice treated with vehicle or PAP-1 (40 mg/kg/die) were stained for GFAP (green; Hoechst, in blue) and visualized at the border of the tumor (white dashed line), scale bar 20 μm. Right, data represent the mean area (in pixels) covered by GFAP + cells present at a distance up to 100 μm from the tumor border (mean ± s.e.m. n = 6, *p = 0.015, unpaired t -test).

    Article Snippet: Immunofluorescence Coronal brain sections (20 μm) were washed in PBS, blocked (3% goat serum in 0.3% Triton X-100) for 1 h at RT and incubated overnight at 4 °C with specific antibodies, GFAP (1:750 –Novus Biologicals, NB300-141), Iba1 (1:750 - Wako, 019-19741), GLT1 (1:1000 – AbCam, ab41621), Kv1.3 (1:100 – Alomone Lab, AGP-005).

    Techniques: Activity Assay, Fluorescence, Size-exclusion Chromatography, Staining, Immunoprecipitation, Cell Culture, Mouse Assay