kv1 3  (Alomone Labs)


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    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: 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
    Price from $9.99 to $1999.99
    kv1 3 - by Bioz Stars, 2021-12
    94/100 stars

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    1) Product Images from "Kv1.3 activity perturbs the homeostatic properties of astrocytes in glioma"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25940-5

    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.
    Figure Legend 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.

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

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

    2) Product Images from "The potassium channel Kv1.3 as a therapeutic target for immunocytoprotection after reperfusion"

    Article Title: The potassium channel Kv1.3 as a therapeutic target for immunocytoprotection after reperfusion

    Journal: Annals of Clinical and Translational Neurology

    doi: 10.1002/acn3.51456

    Comparison of stroke severity in WT versus Kv1.3 −/− mice. (A) Infarct area and deficit score in 16‐week‐old WT ( n = 10) were compared to Kv1.3 −/− ( n = 14) male mice ( p
    Figure Legend Snippet: Comparison of stroke severity in WT versus Kv1.3 −/− mice. (A) Infarct area and deficit score in 16‐week‐old WT ( n = 10) were compared to Kv1.3 −/− ( n = 14) male mice ( p

    Techniques Used: Mouse Assay

    The Kv1.3 blocker PAP‐1 reduces infarction and improves neurological deficit in young and old WT female mice. (A) Infarct area and deficit score in vehicle ( n = 11) were compared to PAP‐1 (40 mg/kg; n = 15) treated 16‐week‐old female mice ( p = 0.007 for infarct, p = 0.016 for NES). (B) Infarct area and deficit score in vehicle ( n = 9) were compared to PAP‐1 (40 mg/kg; n = 9) treated 80‐week‐old female mice ( p = 0.032 for infarct, p = 0.003 for NES). Data are shown as whisker plots with data overlay. The boxes show mean ± SEM and the whiskers show confidence intervals.
    Figure Legend Snippet: The Kv1.3 blocker PAP‐1 reduces infarction and improves neurological deficit in young and old WT female mice. (A) Infarct area and deficit score in vehicle ( n = 11) were compared to PAP‐1 (40 mg/kg; n = 15) treated 16‐week‐old female mice ( p = 0.007 for infarct, p = 0.016 for NES). (B) Infarct area and deficit score in vehicle ( n = 9) were compared to PAP‐1 (40 mg/kg; n = 9) treated 80‐week‐old female mice ( p = 0.032 for infarct, p = 0.003 for NES). Data are shown as whisker plots with data overlay. The boxes show mean ± SEM and the whiskers show confidence intervals.

    Techniques Used: Mouse Assay, Whisker Assay

    Kv1.3 is expressed on microglia/macrophages and T cells in the infarct of an 80‐week‐old female mouse. (A) Sample immunofluorescence staining of 5‐ µ m thick paraffin sections from the 6‐mm slice of an 80‐week‐old female mouse on day 8 after tMCAO. (A) Kv1.3 staining colocalizes to Iba‐1 + cells. (B) Kv1.3 staining on smaller, CD3 + T cells (white arrowheads).
    Figure Legend Snippet: Kv1.3 is expressed on microglia/macrophages and T cells in the infarct of an 80‐week‐old female mouse. (A) Sample immunofluorescence staining of 5‐ µ m thick paraffin sections from the 6‐mm slice of an 80‐week‐old female mouse on day 8 after tMCAO. (A) Kv1.3 staining colocalizes to Iba‐1 + cells. (B) Kv1.3 staining on smaller, CD3 + T cells (white arrowheads).

    Techniques Used: Immunofluorescence, Staining

    PAP‐1 treatment and Kv1.3 deletion reduce Iba‐1 and CD3 staining in 80‐week‐old female mice. Serial paraffin sections (5 µ m thick) cut at 4 mm and the 6 mm from 80‐week‐old female mice were stained for Iba‐1 and CD3. (A) Percentage of Iba‐1‐positive pixels in the infarcted hemisphere. Vehicle‐treated WT ( n = 9), PAP‐1‐treated WT ( n = 9, p = 0.120 for 4‐mm section; p = 0.014 for 6‐mm section), and Kv1.3 −/− ( n = 18, p = 0.011 for 4‐mm section; p = 0.007 for 6‐mm section). (B) Percentage of CD3‐positive pixels in the infarcted hemisphere. Vehicle‐treated WT ( n = 9), PAP‐1‐treated WT ( n = 9, p = 0.049 for 4‐mm section; p = 0.027 for 6‐mm section), and Kv1.3 −/− ( n = 18, p
    Figure Legend Snippet: PAP‐1 treatment and Kv1.3 deletion reduce Iba‐1 and CD3 staining in 80‐week‐old female mice. Serial paraffin sections (5 µ m thick) cut at 4 mm and the 6 mm from 80‐week‐old female mice were stained for Iba‐1 and CD3. (A) Percentage of Iba‐1‐positive pixels in the infarcted hemisphere. Vehicle‐treated WT ( n = 9), PAP‐1‐treated WT ( n = 9, p = 0.120 for 4‐mm section; p = 0.014 for 6‐mm section), and Kv1.3 −/− ( n = 18, p = 0.011 for 4‐mm section; p = 0.007 for 6‐mm section). (B) Percentage of CD3‐positive pixels in the infarcted hemisphere. Vehicle‐treated WT ( n = 9), PAP‐1‐treated WT ( n = 9, p = 0.049 for 4‐mm section; p = 0.027 for 6‐mm section), and Kv1.3 −/− ( n = 18, p

    Techniques Used: Staining, Mouse Assay

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    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: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Comparison of stroke severity in WT versus Kv1.3 −/− mice. (A) Infarct area and deficit score in 16‐week‐old WT ( n = 10) were compared to Kv1.3 −/− ( n = 14) male mice ( p

    Journal: Annals of Clinical and Translational Neurology

    Article Title: The potassium channel Kv1.3 as a therapeutic target for immunocytoprotection after reperfusion

    doi: 10.1002/acn3.51456

    Figure Lengend Snippet: Comparison of stroke severity in WT versus Kv1.3 −/− mice. (A) Infarct area and deficit score in 16‐week‐old WT ( n = 10) were compared to Kv1.3 −/− ( n = 14) male mice ( p

    Article Snippet: The immunofluorescence staining in Figure was performed with a polyclonal guinea pig anti‐Kv1.3 antibody (1:100, AGP‐005 from Alomone Labs) and the same polyclonal rabbit Iba‐1 (1;1000) and CD3 (1:250) antibodies listed above; Secondary Abs: Alexa Fluor® 546‐goat anti‐guinea pig IgG (1:1000, ab150185 from Abcam) and Alexa Fluor®488‐goat anti‐rabbit IgG (1:1500, ab150077 from Abcam).

    Techniques: Mouse Assay

    The Kv1.3 blocker PAP‐1 reduces infarction and improves neurological deficit in young and old WT female mice. (A) Infarct area and deficit score in vehicle ( n = 11) were compared to PAP‐1 (40 mg/kg; n = 15) treated 16‐week‐old female mice ( p = 0.007 for infarct, p = 0.016 for NES). (B) Infarct area and deficit score in vehicle ( n = 9) were compared to PAP‐1 (40 mg/kg; n = 9) treated 80‐week‐old female mice ( p = 0.032 for infarct, p = 0.003 for NES). Data are shown as whisker plots with data overlay. The boxes show mean ± SEM and the whiskers show confidence intervals.

    Journal: Annals of Clinical and Translational Neurology

    Article Title: The potassium channel Kv1.3 as a therapeutic target for immunocytoprotection after reperfusion

    doi: 10.1002/acn3.51456

    Figure Lengend Snippet: The Kv1.3 blocker PAP‐1 reduces infarction and improves neurological deficit in young and old WT female mice. (A) Infarct area and deficit score in vehicle ( n = 11) were compared to PAP‐1 (40 mg/kg; n = 15) treated 16‐week‐old female mice ( p = 0.007 for infarct, p = 0.016 for NES). (B) Infarct area and deficit score in vehicle ( n = 9) were compared to PAP‐1 (40 mg/kg; n = 9) treated 80‐week‐old female mice ( p = 0.032 for infarct, p = 0.003 for NES). Data are shown as whisker plots with data overlay. The boxes show mean ± SEM and the whiskers show confidence intervals.

    Article Snippet: The immunofluorescence staining in Figure was performed with a polyclonal guinea pig anti‐Kv1.3 antibody (1:100, AGP‐005 from Alomone Labs) and the same polyclonal rabbit Iba‐1 (1;1000) and CD3 (1:250) antibodies listed above; Secondary Abs: Alexa Fluor® 546‐goat anti‐guinea pig IgG (1:1000, ab150185 from Abcam) and Alexa Fluor®488‐goat anti‐rabbit IgG (1:1500, ab150077 from Abcam).

    Techniques: Mouse Assay, Whisker Assay

    Kv1.3 is expressed on microglia/macrophages and T cells in the infarct of an 80‐week‐old female mouse. (A) Sample immunofluorescence staining of 5‐ µ m thick paraffin sections from the 6‐mm slice of an 80‐week‐old female mouse on day 8 after tMCAO. (A) Kv1.3 staining colocalizes to Iba‐1 + cells. (B) Kv1.3 staining on smaller, CD3 + T cells (white arrowheads).

    Journal: Annals of Clinical and Translational Neurology

    Article Title: The potassium channel Kv1.3 as a therapeutic target for immunocytoprotection after reperfusion

    doi: 10.1002/acn3.51456

    Figure Lengend Snippet: Kv1.3 is expressed on microglia/macrophages and T cells in the infarct of an 80‐week‐old female mouse. (A) Sample immunofluorescence staining of 5‐ µ m thick paraffin sections from the 6‐mm slice of an 80‐week‐old female mouse on day 8 after tMCAO. (A) Kv1.3 staining colocalizes to Iba‐1 + cells. (B) Kv1.3 staining on smaller, CD3 + T cells (white arrowheads).

    Article Snippet: The immunofluorescence staining in Figure was performed with a polyclonal guinea pig anti‐Kv1.3 antibody (1:100, AGP‐005 from Alomone Labs) and the same polyclonal rabbit Iba‐1 (1;1000) and CD3 (1:250) antibodies listed above; Secondary Abs: Alexa Fluor® 546‐goat anti‐guinea pig IgG (1:1000, ab150185 from Abcam) and Alexa Fluor®488‐goat anti‐rabbit IgG (1:1500, ab150077 from Abcam).

    Techniques: Immunofluorescence, Staining

    PAP‐1 treatment and Kv1.3 deletion reduce Iba‐1 and CD3 staining in 80‐week‐old female mice. Serial paraffin sections (5 µ m thick) cut at 4 mm and the 6 mm from 80‐week‐old female mice were stained for Iba‐1 and CD3. (A) Percentage of Iba‐1‐positive pixels in the infarcted hemisphere. Vehicle‐treated WT ( n = 9), PAP‐1‐treated WT ( n = 9, p = 0.120 for 4‐mm section; p = 0.014 for 6‐mm section), and Kv1.3 −/− ( n = 18, p = 0.011 for 4‐mm section; p = 0.007 for 6‐mm section). (B) Percentage of CD3‐positive pixels in the infarcted hemisphere. Vehicle‐treated WT ( n = 9), PAP‐1‐treated WT ( n = 9, p = 0.049 for 4‐mm section; p = 0.027 for 6‐mm section), and Kv1.3 −/− ( n = 18, p

    Journal: Annals of Clinical and Translational Neurology

    Article Title: The potassium channel Kv1.3 as a therapeutic target for immunocytoprotection after reperfusion

    doi: 10.1002/acn3.51456

    Figure Lengend Snippet: PAP‐1 treatment and Kv1.3 deletion reduce Iba‐1 and CD3 staining in 80‐week‐old female mice. Serial paraffin sections (5 µ m thick) cut at 4 mm and the 6 mm from 80‐week‐old female mice were stained for Iba‐1 and CD3. (A) Percentage of Iba‐1‐positive pixels in the infarcted hemisphere. Vehicle‐treated WT ( n = 9), PAP‐1‐treated WT ( n = 9, p = 0.120 for 4‐mm section; p = 0.014 for 6‐mm section), and Kv1.3 −/− ( n = 18, p = 0.011 for 4‐mm section; p = 0.007 for 6‐mm section). (B) Percentage of CD3‐positive pixels in the infarcted hemisphere. Vehicle‐treated WT ( n = 9), PAP‐1‐treated WT ( n = 9, p = 0.049 for 4‐mm section; p = 0.027 for 6‐mm section), and Kv1.3 −/− ( n = 18, p

    Article Snippet: The immunofluorescence staining in Figure was performed with a polyclonal guinea pig anti‐Kv1.3 antibody (1:100, AGP‐005 from Alomone Labs) and the same polyclonal rabbit Iba‐1 (1;1000) and CD3 (1:250) antibodies listed above; Secondary Abs: Alexa Fluor® 546‐goat anti‐guinea pig IgG (1:1000, ab150185 from Abcam) and Alexa Fluor®488‐goat anti‐rabbit IgG (1:1500, ab150077 from Abcam).

    Techniques: Staining, Mouse Assay