rabbit polyclonal anti kv1 3 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit polyclonal anti kv1 3 antibody
    <t>Kv1.3</t> channels are recruited at the interface between CD3/CD28 beads and T cells
    Rabbit Polyclonal Anti Kv1 3 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti kv1 3 antibody/product/Alomone Labs
    Average 93 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti kv1 3 antibody - by Bioz Stars, 2022-08
    93/100 stars

    Images

    1) Product Images from "ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1 , 2"

    Article Title: ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1 , 2

    Journal:

    doi:

    Kv1.3 channels are recruited at the interface between CD3/CD28 beads and T cells
    Figure Legend Snippet: Kv1.3 channels are recruited at the interface between CD3/CD28 beads and T cells

    Techniques Used:

    Kv1.3 channels in T lymphocytes from patients with SLE display biophysical and pharmacological properties similar to those in healthy T cells
    Figure Legend Snippet: Kv1.3 channels in T lymphocytes from patients with SLE display biophysical and pharmacological properties similar to those in healthy T cells

    Techniques Used:

    Comparison of the rates of Kv1.3 channel compartmentalization in the IS in normal and SLE T cells
    Figure Legend Snippet: Comparison of the rates of Kv1.3 channel compartmentalization in the IS in normal and SLE T cells

    Techniques Used:

    Electrophysiological and pharmacological properties of Kv1.3 channels in SLE T cells
    Figure Legend Snippet: Electrophysiological and pharmacological properties of Kv1.3 channels in SLE T cells

    Techniques Used:

    APC-T cell activation induces differential reorganization of Kv1.3 channels in the IS formed with resting healthy and SLE T cells
    Figure Legend Snippet: APC-T cell activation induces differential reorganization of Kv1.3 channels in the IS formed with resting healthy and SLE T cells

    Techniques Used: Activation Assay

    Differential kinetics of Kv1.3 channel reorganization in the IS
    Figure Legend Snippet: Differential kinetics of Kv1.3 channel reorganization in the IS

    Techniques Used:

    Kv1.3 channel recruitment in the IS in activated healthy T cells parallels SLE T lymphocytes
    Figure Legend Snippet: Kv1.3 channel recruitment in the IS in activated healthy T cells parallels SLE T lymphocytes

    Techniques Used:

    The kinetics of Kv1.3 redistribution in the immunological synapse of SLE T cells resemble those of pre-activated normal T cells
    Figure Legend Snippet: The kinetics of Kv1.3 redistribution in the immunological synapse of SLE T cells resemble those of pre-activated normal T cells

    Techniques Used:

    2) Product Images from "Model Senescent Microglia Induce Disease Related Changes in α-Synuclein Expression and Activity"

    Article Title: Model Senescent Microglia Induce Disease Related Changes in α-Synuclein Expression and Activity

    Journal: Biomolecules

    doi: 10.3390/biom8030067

    Expression of the potassium channel Kv1.3. Protein extracts were prepared from control and iron-fed C8B4 microglia. Western blot analysis was carried out to determine the level of Kv1.3 in the microglia. Bands for the protein were observed in both control and iron-fed microglia. Levels of tubulin were also determined to verify protein loading. The results showed a significant ( p
    Figure Legend Snippet: Expression of the potassium channel Kv1.3. Protein extracts were prepared from control and iron-fed C8B4 microglia. Western blot analysis was carried out to determine the level of Kv1.3 in the microglia. Bands for the protein were observed in both control and iron-fed microglia. Levels of tubulin were also determined to verify protein loading. The results showed a significant ( p

    Techniques Used: Expressing, Western Blot

    3) Product Images from "Neurotrophin modulation of voltage-gated potassium channels in rat through TrkB receptors is time and sensory experience dependent"

    Article Title: Neurotrophin modulation of voltage-gated potassium channels in rat through TrkB receptors is time and sensory experience dependent

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2002.017376

    Recognition of Kv1.3 protein by α-AU13 antiserum by Western blot analysis Cell lysates from Kv1.3 transfected HEK293 cells, separated by SDS-PAGE and visualized by Western blot analysis with various dilutions of the antiserum from 1 : 500 to 1:10 000.
    Figure Legend Snippet: Recognition of Kv1.3 protein by α-AU13 antiserum by Western blot analysis Cell lysates from Kv1.3 transfected HEK293 cells, separated by SDS-PAGE and visualized by Western blot analysis with various dilutions of the antiserum from 1 : 500 to 1:10 000.

    Techniques Used: Western Blot, Transfection, SDS Page

    Sensory deprivation by unilateral naris occlusion alters BDNF-stimulated tyrosine phosphorylation of Kv1.3 channel P1 animals were left naris occluded by cauterization and raised with odour sensory deprivation to this naris from P20 to P25. A , olfactory bulbs contralateral (non-occluded) and ipsilateral (occluded) to the cauterized naris were then harvested, stimulated with BDNF and immunoprecipitated with anti-Kv1.3 and blotted with anti-4G10. Total tyrosine phosphorylation of Kv1.3 is indicated by the arrow. B , histogram of the mean increase in tyrosine phosphorylation of Kv1.3 by acute BDNF stimulation comparing non-occluded versus sensory-deprived conditions (occluded). Pixel values were calculated by quantitative densitometry. The difference in pixel density between unstimulated and BDNF-stimulated olfactory bulbs was plotted for occluded and non-occluded naris conditions, where 0 = no change in Kv1.3 phosphorylation with BDNF treatment. * Significantly different by Student's paired t test, P
    Figure Legend Snippet: Sensory deprivation by unilateral naris occlusion alters BDNF-stimulated tyrosine phosphorylation of Kv1.3 channel P1 animals were left naris occluded by cauterization and raised with odour sensory deprivation to this naris from P20 to P25. A , olfactory bulbs contralateral (non-occluded) and ipsilateral (occluded) to the cauterized naris were then harvested, stimulated with BDNF and immunoprecipitated with anti-Kv1.3 and blotted with anti-4G10. Total tyrosine phosphorylation of Kv1.3 is indicated by the arrow. B , histogram of the mean increase in tyrosine phosphorylation of Kv1.3 by acute BDNF stimulation comparing non-occluded versus sensory-deprived conditions (occluded). Pixel values were calculated by quantitative densitometry. The difference in pixel density between unstimulated and BDNF-stimulated olfactory bulbs was plotted for occluded and non-occluded naris conditions, where 0 = no change in Kv1.3 phosphorylation with BDNF treatment. * Significantly different by Student's paired t test, P

    Techniques Used: Immunoprecipitation

    Acute BDNF stimulation increases the tyrosine phosphorylation of Kv1.3 in the rat olfactory bulb A , olfactory bulbs were stimulated with or without 100 ng ml −1 of BDNF in PBS for 20 min, homogenized, immunoprecipitated with anti-phosphotyrosine antiserum (anti-4G10), separated by SDS-PAGE, and Western blots were probed with anti-Kv1.3 (α-AU13). Total tyrosine phosphorylation of Kv1.3 is shown in the bottom panel. The heavy chain of IgG is also indicated below that of Kv1.3. Ten micrograms of cell lysate were blotted for α-AU13 and α-TrkB, respectively, to confirm equivalent protein expression of the channel and receptor under BDNF-stimulated and unstimulated conditions (top panels). B , histogram of quantitative densitometry of four experiments as in A . Mean pixel density of Kv1.3 tyrosine phosphorylation under control versus BDNF-stimulated conditions. * Significantly different, Arc Sin transformation for percentile data, Student's t test P
    Figure Legend Snippet: Acute BDNF stimulation increases the tyrosine phosphorylation of Kv1.3 in the rat olfactory bulb A , olfactory bulbs were stimulated with or without 100 ng ml −1 of BDNF in PBS for 20 min, homogenized, immunoprecipitated with anti-phosphotyrosine antiserum (anti-4G10), separated by SDS-PAGE, and Western blots were probed with anti-Kv1.3 (α-AU13). Total tyrosine phosphorylation of Kv1.3 is shown in the bottom panel. The heavy chain of IgG is also indicated below that of Kv1.3. Ten micrograms of cell lysate were blotted for α-AU13 and α-TrkB, respectively, to confirm equivalent protein expression of the channel and receptor under BDNF-stimulated and unstimulated conditions (top panels). B , histogram of quantitative densitometry of four experiments as in A . Mean pixel density of Kv1.3 tyrosine phosphorylation under control versus BDNF-stimulated conditions. * Significantly different, Arc Sin transformation for percentile data, Student's t test P

    Techniques Used: Immunoprecipitation, SDS Page, Western Blot, Expressing, Transformation Assay

    4) Product Images from "Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease"

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI136174

    Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
    Figure Legend Snippet: Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

    Techniques Used: Modification, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Immunohistochemistry, Mouse Assay, Injection, Inhibition, Transfection, Plasmid Preparation, Quantitative RT-PCR, Griess Assay, Luminex

    Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P
    Figure Legend Snippet: Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P

    Techniques Used: Inhibition, Activity Assay, Mouse Assay, High Performance Liquid Chromatography, Western Blot

    Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P
    Figure Legend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P

    Techniques Used: Expressing, Ex Vivo, Derivative Assay, Quantitative RT-PCR, Western Blot, Isolation, Activity Assay

    Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
    Figure Legend Snippet: Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Mouse Assay, Immunohistochemistry, Immunostaining, Staining

    Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
    Figure Legend Snippet: Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Techniques Used: In Silico, Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot, Patch Clamp, Activity Assay, Immunocytochemistry

    Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
    Figure Legend Snippet: Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

    Techniques Used: Cell Culture, Luminex, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Lactate Dehydrogenase Assay, Western Blot, Expressing, Immunocytochemistry

    Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P
    Figure Legend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P

    Techniques Used: Expressing, In Vitro, Patch Clamp, Activity Assay, Quantitative RT-PCR, Western Blot, Immunocytochemistry

    5) Product Images from "Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease"

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI136174

    Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
    Figure Legend Snippet: Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

    Techniques Used: Modification, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Immunohistochemistry, Mouse Assay, Injection, Inhibition, Transfection, Plasmid Preparation, Quantitative RT-PCR, Griess Assay, Luminex

    Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P
    Figure Legend Snippet: Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P

    Techniques Used: Inhibition, Activity Assay, Mouse Assay, High Performance Liquid Chromatography, Western Blot

    Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P
    Figure Legend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P

    Techniques Used: Expressing, Ex Vivo, Derivative Assay, Quantitative RT-PCR, Western Blot, Isolation, Activity Assay

    Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
    Figure Legend Snippet: Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Mouse Assay, Immunohistochemistry, Immunostaining, Staining

    Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
    Figure Legend Snippet: Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Techniques Used: In Silico, Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot, Patch Clamp, Activity Assay, Immunocytochemistry

    Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
    Figure Legend Snippet: Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

    Techniques Used: Cell Culture, Luminex, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Lactate Dehydrogenase Assay, Western Blot, Expressing, Immunocytochemistry

    Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P
    Figure Legend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P

    Techniques Used: Expressing, In Vitro, Patch Clamp, Activity Assay, Quantitative RT-PCR, Western Blot, Immunocytochemistry

    6) Product Images from "Kv1.3 channels regulate synaptic transmission in the nucleus of solitary tract"

    Article Title: Kv1.3 channels regulate synaptic transmission in the nucleus of solitary tract

    Journal: Journal of Neurophysiology

    doi: 10.1152/jn.00494.2010

    Immunohistochemical localization of Kv1.3 in the NG and aortic depressor nerve (ADN). A : anti-Kv1.3 (Neuromab) immunolabeling in postnatal day 30 Spraque-Dawley rat nodose slices. The image is a mass z -projection of five confocal sections acquired at
    Figure Legend Snippet: Immunohistochemical localization of Kv1.3 in the NG and aortic depressor nerve (ADN). A : anti-Kv1.3 (Neuromab) immunolabeling in postnatal day 30 Spraque-Dawley rat nodose slices. The image is a mass z -projection of five confocal sections acquired at

    Techniques Used: Immunohistochemistry, Immunolabeling

    Detection of Kv1.3 α-subunits. A : PCR products resulting from the amplification of first-strand cDNA prepared with (+) or without (−) reverse transcriptase (RT) from the rat nodose ganglia (NG) or brain poly-A+ RNA with Kv1.3-specific
    Figure Legend Snippet: Detection of Kv1.3 α-subunits. A : PCR products resulting from the amplification of first-strand cDNA prepared with (+) or without (−) reverse transcriptase (RT) from the rat nodose ganglia (NG) or brain poly-A+ RNA with Kv1.3-specific

    Techniques Used: Polymerase Chain Reaction, Amplification

    Confocal images of the NTS labeled with Kv1.3. A : confocal image of a horizontal NTS section in the commissural region. Fine fibers in the solitary tract (TS) exit the tract and innervate neurons medial to the tract. B : confocal image of a horizontal
    Figure Legend Snippet: Confocal images of the NTS labeled with Kv1.3. A : confocal image of a horizontal NTS section in the commissural region. Fine fibers in the solitary tract (TS) exit the tract and innervate neurons medial to the tract. B : confocal image of a horizontal

    Techniques Used: Labeling

    7) Product Images from "Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation *"

    Article Title: Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.678995

    Characterization of the effects on proliferation of Kv1.3 COOH terminus point mutants. The schematic shows the position in the C terminus of Kv1.3 of the phosphorylatable residues (in red ) that have been mutated to alanine. Also, AMP kinase and the insulin receptor kinase putative motifs are indicated as well as the YS segment. Conserved residues in the C terminus of Kv1.3 are underlined . The upper panel shows EdU incorporation assay of HEK293 cells transfected with each point mutant channels using Cherry and Kv1.3 expressing cells as controls. Each bar is the mean ± S.E. ( n = 6–20 determinations from 3–7 different experiments). *, p
    Figure Legend Snippet: Characterization of the effects on proliferation of Kv1.3 COOH terminus point mutants. The schematic shows the position in the C terminus of Kv1.3 of the phosphorylatable residues (in red ) that have been mutated to alanine. Also, AMP kinase and the insulin receptor kinase putative motifs are indicated as well as the YS segment. Conserved residues in the C terminus of Kv1.3 are underlined . The upper panel shows EdU incorporation assay of HEK293 cells transfected with each point mutant channels using Cherry and Kv1.3 expressing cells as controls. Each bar is the mean ± S.E. ( n = 6–20 determinations from 3–7 different experiments). *, p

    Techniques Used: Transfection, Mutagenesis, Expressing

    Characterization of the Kv1.3 and Kv1.5 mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).
    Figure Legend Snippet: Characterization of the Kv1.3 and Kv1.5 mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).

    Techniques Used: Mutagenesis, Activation Assay, Transfection, Staining, Expressing

    8) Product Images from "Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression"

    Article Title: Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression

    Journal: Cancers

    doi: 10.3390/cancers13174457

    Kv1.3 accumulates at perinuclear mitochondria during the G1/S transition. ( A ) Subcellular fractionation of 3T3-L1 wild-type preadipocytes to obtain the membranous (Mb) and mitochondrial (Mit) fractions. The samples were probed for Kv1.3, Na+/K+ ATPase (a membrane marker) and TIMM50 (a mitochondrial marker). ( B ) Electron micrograph showing mitochondria of 3T3-L1 wild-type preadipocytes. Kv1.3 was labeled with 18 nm immunogold particles (black arrowhead) and was located at the inner mitochondrial membrane. The scale bar represents 200 nm. ( C – H ) Cells were either in the G0/G1 or the G1/S phase following serum deprivation or serum readdition for 12 h, respectively. Representative confocal images showing Kv1.3 and mitochondria in wild-type preadipocytes fixed in the G0/G1 ( C – E ) and G1/S ( F – H ) phase. Ea-Eb and Ha-Hb are magnified images of E and H, respectively. Ea and Ha show distal regions, and Eb and Hb show perinuclear regions. Yellow indicates colocalization of Kv1.3 (green) and mitochondria (red). The scale bar represents 20 µm. ( I ) Pearson’s coefficient of colocalization between Kv1.3 and mitochondria. The data are the mean ± SE ( n > 30). *** p
    Figure Legend Snippet: Kv1.3 accumulates at perinuclear mitochondria during the G1/S transition. ( A ) Subcellular fractionation of 3T3-L1 wild-type preadipocytes to obtain the membranous (Mb) and mitochondrial (Mit) fractions. The samples were probed for Kv1.3, Na+/K+ ATPase (a membrane marker) and TIMM50 (a mitochondrial marker). ( B ) Electron micrograph showing mitochondria of 3T3-L1 wild-type preadipocytes. Kv1.3 was labeled with 18 nm immunogold particles (black arrowhead) and was located at the inner mitochondrial membrane. The scale bar represents 200 nm. ( C – H ) Cells were either in the G0/G1 or the G1/S phase following serum deprivation or serum readdition for 12 h, respectively. Representative confocal images showing Kv1.3 and mitochondria in wild-type preadipocytes fixed in the G0/G1 ( C – E ) and G1/S ( F – H ) phase. Ea-Eb and Ha-Hb are magnified images of E and H, respectively. Ea and Ha show distal regions, and Eb and Hb show perinuclear regions. Yellow indicates colocalization of Kv1.3 (green) and mitochondria (red). The scale bar represents 20 µm. ( I ) Pearson’s coefficient of colocalization between Kv1.3 and mitochondria. The data are the mean ± SE ( n > 30). *** p

    Techniques Used: Fractionation, Marker, Labeling

    Kv1.3 regulates the mitochondrial membrane potential during the cell cycle. Ablation of Kv1.3 impairs the mitochondrial membrane potential. ( A ) TMRM intensity in wild-type (black bar) and Kv1.3KD (white bar) 3T3-L1 preadipocytes was analyzed with flow cytometry. The data are the mean ± SE ( n = 3), * p
    Figure Legend Snippet: Kv1.3 regulates the mitochondrial membrane potential during the cell cycle. Ablation of Kv1.3 impairs the mitochondrial membrane potential. ( A ) TMRM intensity in wild-type (black bar) and Kv1.3KD (white bar) 3T3-L1 preadipocytes was analyzed with flow cytometry. The data are the mean ± SE ( n = 3), * p

    Techniques Used: Flow Cytometry

    Kv1.3 facilitates the G1/S transition of the cell cycle in preadipocytes. Serum-starved resting cells were incubated for the indicated time after serum readdition. ( A ) Cell cycle analysis of 3T3-L1 preadipocytes was performed with propidium iodide. Representative histograms at 0, 6, 12, 18 or 24 h after serum readdition. The cells exhibit two blue peaks corresponding to the G0/G1 (left) and G2 (right) phases. The cell population in purple corresponds to cells in the S phase. Left panels, wild-type preadipocytes; right panels, Kv1.3KD preadipocytes. ( B ) The % of cells in the G0/G1 phase, % of cells in the S phase and % of cells in the G2 phase for wild-type (black) and Kv1.3KD (white) preadipocytes. The data are the mean ± SE ( n = 4–10 independent experiments). Two-way ANOVA indicated p
    Figure Legend Snippet: Kv1.3 facilitates the G1/S transition of the cell cycle in preadipocytes. Serum-starved resting cells were incubated for the indicated time after serum readdition. ( A ) Cell cycle analysis of 3T3-L1 preadipocytes was performed with propidium iodide. Representative histograms at 0, 6, 12, 18 or 24 h after serum readdition. The cells exhibit two blue peaks corresponding to the G0/G1 (left) and G2 (right) phases. The cell population in purple corresponds to cells in the S phase. Left panels, wild-type preadipocytes; right panels, Kv1.3KD preadipocytes. ( B ) The % of cells in the G0/G1 phase, % of cells in the S phase and % of cells in the G2 phase for wild-type (black) and Kv1.3KD (white) preadipocytes. The data are the mean ± SE ( n = 4–10 independent experiments). Two-way ANOVA indicated p

    Techniques Used: Incubation, Cell Cycle Assay

    Representative cartoon summarizing the participation of the mitochondrial Kv1.3 (mitoKv1.3) in the proliferation of preadipocytes. Kv1.3 would facilitate the G1/S transition of the cell cycle in preadipocytes accumulating at perinuclear mitochondria. The elucidation of a putative mitochondrial-nuclear communication during this phase of the cell cycle in which Kv1.3 would participate deserves much effort. During the G1/S transition, Kv1.3 would contribute to the mitochondrial fusion/fission equilibrium controlling the mitochondrial membrane potential. Ablation of Kv1.3 (Kv1.3KD) would impair mitochondrial dynamics during cell cycle progression. Kv1.3KD, 3T3-L1 preadipocytes, with a genetic ablation of Kv1.3. Green dots, Kv1.3 channels; magenta, mitochondrial network.
    Figure Legend Snippet: Representative cartoon summarizing the participation of the mitochondrial Kv1.3 (mitoKv1.3) in the proliferation of preadipocytes. Kv1.3 would facilitate the G1/S transition of the cell cycle in preadipocytes accumulating at perinuclear mitochondria. The elucidation of a putative mitochondrial-nuclear communication during this phase of the cell cycle in which Kv1.3 would participate deserves much effort. During the G1/S transition, Kv1.3 would contribute to the mitochondrial fusion/fission equilibrium controlling the mitochondrial membrane potential. Ablation of Kv1.3 (Kv1.3KD) would impair mitochondrial dynamics during cell cycle progression. Kv1.3KD, 3T3-L1 preadipocytes, with a genetic ablation of Kv1.3. Green dots, Kv1.3 channels; magenta, mitochondrial network.

    Techniques Used:

    Kv1.3 participates in the proliferation of preadipocytes. 3T3-L1 preadipocytes express Kv1.3, and genetic ablation of the channel alters cell proliferation. ( A ) Representative immunofluorescence confocal image of Kv1.3 in 3T3-L1 preadipocytes. The scale bar represents 20 µm. ( B ) Kv1.3 silencing in 3T3-L1 preadipocytes. Cells were infected with Kv1.3 shRNA (Kv1.3KD) or scramble shRNA (SCR) lentivirus. β-actin was used as a loading control. Noninfected 3T3-L1 cells were called wild-type (WT) cells. ( C ) Quantification of the efficiency of Kv1.3 silencing. The data are the mean ± SE ( n ≥ 3). * p
    Figure Legend Snippet: Kv1.3 participates in the proliferation of preadipocytes. 3T3-L1 preadipocytes express Kv1.3, and genetic ablation of the channel alters cell proliferation. ( A ) Representative immunofluorescence confocal image of Kv1.3 in 3T3-L1 preadipocytes. The scale bar represents 20 µm. ( B ) Kv1.3 silencing in 3T3-L1 preadipocytes. Cells were infected with Kv1.3 shRNA (Kv1.3KD) or scramble shRNA (SCR) lentivirus. β-actin was used as a loading control. Noninfected 3T3-L1 cells were called wild-type (WT) cells. ( C ) Quantification of the efficiency of Kv1.3 silencing. The data are the mean ± SE ( n ≥ 3). * p

    Techniques Used: Immunofluorescence, Infection, shRNA

    Kv1.3 regulates the mitochondrial fusion/fission equilibrium during the G1/S transition. Confocal images showing mitochondria in cells fixed in the G0/G1 ( A – F ) and G1/S ( G – L ) phase for WT ( A – C , G – I ) and Kv1.3KD preadipocytes ( D – F , J – L ). The scale bar represents 20 µm. Images were processed (tubeness and skeleton) to perform morphometric analysis of mitochondria. ( M ) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. ( N ) Number of mitochondrial particles per µm 2 . ( O ) The form factor describes the particle shape complexity and was computed as the average (perimeter)2/(4π·area). A circle corresponds to a minimum value of 1. The data are the mean ± SE ( n > 30). *, p
    Figure Legend Snippet: Kv1.3 regulates the mitochondrial fusion/fission equilibrium during the G1/S transition. Confocal images showing mitochondria in cells fixed in the G0/G1 ( A – F ) and G1/S ( G – L ) phase for WT ( A – C , G – I ) and Kv1.3KD preadipocytes ( D – F , J – L ). The scale bar represents 20 µm. Images were processed (tubeness and skeleton) to perform morphometric analysis of mitochondria. ( M ) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. ( N ) Number of mitochondrial particles per µm 2 . ( O ) The form factor describes the particle shape complexity and was computed as the average (perimeter)2/(4π·area). A circle corresponds to a minimum value of 1. The data are the mean ± SE ( n > 30). *, p

    Techniques Used:

    9) Product Images from "Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4‐mediated calcium entry in microglia, et al. Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4‐mediated calcium entry in microglia"

    Article Title: Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4‐mediated calcium entry in microglia, et al. Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4‐mediated calcium entry in microglia

    Journal: Glia

    doi: 10.1002/glia.23847

    Kv1.3 channel inhibition reduces intracellular Ca 2+ signaling. (a) Fluo‐4 AM calcium indicator fluorescence signal elicited by 0.1 mM ATP is 2.65 ± 0.99‐fold ( n = 3) higher in total area under the curve (AUC) compared to that elicited by 0.1 mM BzATP ( n = 3). Statistical significance determined by unpaired t test comparing ATP and BzATP cells. (b) Ivermectin (IVC, 3 μM) increases fluorescence signaling elicited by 0.1 mM ATP by 2.65 ± 0.99‐fold ( n = 4). Statistical significance between before and after ivermectin determined by paired t test. (c) Twenty‐four hours treatment with lipopolysaccharides (LPS) (300 ng/ml) or interleukin‐4 (IL‐4) (20 ng/ml) suppresses fluorescence increase. Statistical significance determined by one‐way analysis of variance (ANOVA) followed by Tukey–Cramer's post hoc test (alpha = 0.05). (d–f) Preincubation with the Kv1.3 blocker ShK‐223 (100 nM) reduces ATP‐mediated fluorescence increases in LPS‐treated microglia but not in undifferentiated or IL‐4 stimulated microglia. All ATP applied at 0.1 mM and after baseline fluorescence was recorded for 2 min. Changes in [Ca 2+ ] i are represented as ΔF/F (change in fluorescence measured as AUC after baseline subtraction). Scale bars indicate 20% of the maximal normalized change in ΔF/F, which is 1ΔF/F. Statistical significance determined by paired t test. All data presented as mean ± SEM . Measurements from three to seven separate experiments (coverslips from different cultures on different days) and 50–100 cells each were measured per experiment for panels (c)–(f). * p
    Figure Legend Snippet: Kv1.3 channel inhibition reduces intracellular Ca 2+ signaling. (a) Fluo‐4 AM calcium indicator fluorescence signal elicited by 0.1 mM ATP is 2.65 ± 0.99‐fold ( n = 3) higher in total area under the curve (AUC) compared to that elicited by 0.1 mM BzATP ( n = 3). Statistical significance determined by unpaired t test comparing ATP and BzATP cells. (b) Ivermectin (IVC, 3 μM) increases fluorescence signaling elicited by 0.1 mM ATP by 2.65 ± 0.99‐fold ( n = 4). Statistical significance between before and after ivermectin determined by paired t test. (c) Twenty‐four hours treatment with lipopolysaccharides (LPS) (300 ng/ml) or interleukin‐4 (IL‐4) (20 ng/ml) suppresses fluorescence increase. Statistical significance determined by one‐way analysis of variance (ANOVA) followed by Tukey–Cramer's post hoc test (alpha = 0.05). (d–f) Preincubation with the Kv1.3 blocker ShK‐223 (100 nM) reduces ATP‐mediated fluorescence increases in LPS‐treated microglia but not in undifferentiated or IL‐4 stimulated microglia. All ATP applied at 0.1 mM and after baseline fluorescence was recorded for 2 min. Changes in [Ca 2+ ] i are represented as ΔF/F (change in fluorescence measured as AUC after baseline subtraction). Scale bars indicate 20% of the maximal normalized change in ΔF/F, which is 1ΔF/F. Statistical significance determined by paired t test. All data presented as mean ± SEM . Measurements from three to seven separate experiments (coverslips from different cultures on different days) and 50–100 cells each were measured per experiment for panels (c)–(f). * p

    Techniques Used: Inhibition, Fluorescence

    Kv1.3 prevents extreme membrane depolarization triggered by current injections. Sample current‐clamp traces of a Kv1.3+ Chinese Hamster Ovary (CHO) cell before (a) and after (b) 100 nM ShK‐223 ( n = 8). (Top) Current injection protocol consisted of 3‐s ramps from 0 to 50 pA in 10 pA steps. Insets : Voltage‐clamp traces of same cell elicited by a voltage ramp from −120 to +40 mV. (c) Quantification of maximal membrane depolarization measured. Sample current‐clamp traces of Kv1.3+ microglia before (d) and after (e) 100 nM ShK‐223 ( n = 11). (Top) Current injection protocol consisted of 3‐s ramps from 0 to 25 pA in 5 pA steps. Insets : Voltage‐clamp traces of same cell. (f) Quantification of maximal membrane depolarization measured. Dashed green lines indicate the −40‐mV membrane potential level near the Kv1.3 activation threshold potential. Error bars indicate mean ± SD . Statistical significance determined by paired t test. * p
    Figure Legend Snippet: Kv1.3 prevents extreme membrane depolarization triggered by current injections. Sample current‐clamp traces of a Kv1.3+ Chinese Hamster Ovary (CHO) cell before (a) and after (b) 100 nM ShK‐223 ( n = 8). (Top) Current injection protocol consisted of 3‐s ramps from 0 to 50 pA in 10 pA steps. Insets : Voltage‐clamp traces of same cell elicited by a voltage ramp from −120 to +40 mV. (c) Quantification of maximal membrane depolarization measured. Sample current‐clamp traces of Kv1.3+ microglia before (d) and after (e) 100 nM ShK‐223 ( n = 11). (Top) Current injection protocol consisted of 3‐s ramps from 0 to 25 pA in 5 pA steps. Insets : Voltage‐clamp traces of same cell. (f) Quantification of maximal membrane depolarization measured. Dashed green lines indicate the −40‐mV membrane potential level near the Kv1.3 activation threshold potential. Error bars indicate mean ± SD . Statistical significance determined by paired t test. * p

    Techniques Used: Injection, Activation Assay

    Effects of Kv1.3 channel inhibition on mRNA expression of microglial channels and pro‐inflammatory cytokines. Quantitative PCR ( qPCR) quantification of mRNA expression of (a) channels and (b) microglia‐associated cytokines in undifferentiated (U), lipopolysaccharides (LPS) only (L; 300 ng/ml), LPS + 100 nM ShK‐223 (L + S) and 100 nM ShK‐223 only (S) treated microglia. Data from three independent mixed‐gender postnatal microglia cultures. Bar graphs represent means ± SEM . Statistical analysis was performed using unpaired t test. * p
    Figure Legend Snippet: Effects of Kv1.3 channel inhibition on mRNA expression of microglial channels and pro‐inflammatory cytokines. Quantitative PCR ( qPCR) quantification of mRNA expression of (a) channels and (b) microglia‐associated cytokines in undifferentiated (U), lipopolysaccharides (LPS) only (L; 300 ng/ml), LPS + 100 nM ShK‐223 (L + S) and 100 nM ShK‐223 only (S) treated microglia. Data from three independent mixed‐gender postnatal microglia cultures. Bar graphs represent means ± SEM . Statistical analysis was performed using unpaired t test. * p

    Techniques Used: Inhibition, Expressing, Real-time Polymerase Chain Reaction

    Influence of Kv1.3 expression on membrane potential changes. (a) Corresponding voltage‐clamp ( top ) and current‐clamp ( bottom ) traces induced by 0.1 mM ATP from three individual microglia. (b) Scatterplots for resting membrane potential (RMP) and ATP‐induced membrane potential (AMP). RMP's measured in undifferentiated cells and lipopolysaccharides (LPS)‐differentiated cells averaged −88.08 ± 5.14 mV ( n = 27) and −67.64 ± 12.62 mV ( n = 28), respectively. AMP's in undifferentiated cells and LPS‐differentiated cells averaged −19.32 ± 14.49 mV and −44.09 ± 7.67 mV, respectively. Data represented by means ± SD . Statistical significance between before and after ATP addition determined by paired t test and between undifferentiated and lipopolysaccharides (LPS)‐differentiated microglia determined by one‐way analysis of variance (ANOVA) followed by post hoc Tukey–Cramer's test. *** p
    Figure Legend Snippet: Influence of Kv1.3 expression on membrane potential changes. (a) Corresponding voltage‐clamp ( top ) and current‐clamp ( bottom ) traces induced by 0.1 mM ATP from three individual microglia. (b) Scatterplots for resting membrane potential (RMP) and ATP‐induced membrane potential (AMP). RMP's measured in undifferentiated cells and lipopolysaccharides (LPS)‐differentiated cells averaged −88.08 ± 5.14 mV ( n = 27) and −67.64 ± 12.62 mV ( n = 28), respectively. AMP's in undifferentiated cells and LPS‐differentiated cells averaged −19.32 ± 14.49 mV and −44.09 ± 7.67 mV, respectively. Data represented by means ± SD . Statistical significance between before and after ATP addition determined by paired t test and between undifferentiated and lipopolysaccharides (LPS)‐differentiated microglia determined by one‐way analysis of variance (ANOVA) followed by post hoc Tukey–Cramer's test. *** p

    Techniques Used: Expressing

    Expression changes of Kv1.3 channels and P2X4 receptors in in microglia isolated from Cx3CR1 +/EGFP transgenic mice 8 days after middle cerebral artery occlusion (MCAO) as a model of ischemic stroke. Sample immunofluorescence staining of 14‐μM thick coronal brain sections from the 6‐mm depth showing (a) increased Kv1.3 ( red ) and (b) P2X4 ( red ) immunoreactivity in ipsilateral Cx3CR1 +/EGFP ( green ) cells but not contralateral cells. Each channel was analyzed on n = 3–4 coronal sections from N = 3 male and 3 female mice. (c) P2X4 (d) Kv1.3 and (e) Kir2.1 current densities measured from CD11b + Cx3CR1 +/EGFP microglia acutely isolated from the ipsilateral hemisphere (8 days after MCAO) compared to microglia isolated from the contralateral side. Statistical significance determined by one‐way analysis of variance (ANOVA) followed by Tukey–Cramer's post hoc (alpha = 0.05). * p
    Figure Legend Snippet: Expression changes of Kv1.3 channels and P2X4 receptors in in microglia isolated from Cx3CR1 +/EGFP transgenic mice 8 days after middle cerebral artery occlusion (MCAO) as a model of ischemic stroke. Sample immunofluorescence staining of 14‐μM thick coronal brain sections from the 6‐mm depth showing (a) increased Kv1.3 ( red ) and (b) P2X4 ( red ) immunoreactivity in ipsilateral Cx3CR1 +/EGFP ( green ) cells but not contralateral cells. Each channel was analyzed on n = 3–4 coronal sections from N = 3 male and 3 female mice. (c) P2X4 (d) Kv1.3 and (e) Kir2.1 current densities measured from CD11b + Cx3CR1 +/EGFP microglia acutely isolated from the ipsilateral hemisphere (8 days after MCAO) compared to microglia isolated from the contralateral side. Statistical significance determined by one‐way analysis of variance (ANOVA) followed by Tukey–Cramer's post hoc (alpha = 0.05). * p

    Techniques Used: Expressing, Isolation, Transgenic Assay, Mouse Assay, Immunofluorescence, Staining

    Kv1.3 blockade depolarizes microglia and disrupts resistance to ATP‐induced membrane depolarization. (a) Kv1.3 inhibitors do not cross‐react with P2X4. Sample recording of P2X4 currents elicited by 0.1 mM ATP in a Chinese Hamster Ovary (CHO) cell at the 0, 5, and 10‐min time points displaying characteristic time‐dependent current rundown. (b) Bar graphs showing normalized current for control cells ( n = 5), PAP‐1 (1 μM) treated cells ( n = 4), and ShK‐223 (100 nM) treated cells ( n = 5). Inhibitors were added immediately after the first ATP pulse and remained in the recording chamber throughout the duration between and during subsequent ATP pulses. Error bars denote means ± SD . (c) Voltage‐clamp currents before and after inhibition of Kv1.3 with 100 nM ShK‐223 in an undifferentiated microglial cell. (g) Current‐clamp displaying ATP‐induced depolarization (AID) of resting membrane potential (RMP) before and after ShK‐223 in the same undifferentiated cell. (e) Scatterplots summarizing RMP and AMP levels before and after ShK‐223 for undifferentiated cells ( n = 14). (f) Voltage‐clamp currents before and after inhibition of Kv1.3 with 100 nM ShK‐223 in an lipopolysaccharides (LPS)‐stimulated microglial cell. (g) Current‐clamp displaying AID of RMP before and after ShK‐223 in the same LPS‐stimulated cell. (h) Scatterplots summarizing RMP and AMP levels for LPS‐treated cells ( n = 8). Statistical significance determined by paired t test. *** p
    Figure Legend Snippet: Kv1.3 blockade depolarizes microglia and disrupts resistance to ATP‐induced membrane depolarization. (a) Kv1.3 inhibitors do not cross‐react with P2X4. Sample recording of P2X4 currents elicited by 0.1 mM ATP in a Chinese Hamster Ovary (CHO) cell at the 0, 5, and 10‐min time points displaying characteristic time‐dependent current rundown. (b) Bar graphs showing normalized current for control cells ( n = 5), PAP‐1 (1 μM) treated cells ( n = 4), and ShK‐223 (100 nM) treated cells ( n = 5). Inhibitors were added immediately after the first ATP pulse and remained in the recording chamber throughout the duration between and during subsequent ATP pulses. Error bars denote means ± SD . (c) Voltage‐clamp currents before and after inhibition of Kv1.3 with 100 nM ShK‐223 in an undifferentiated microglial cell. (g) Current‐clamp displaying ATP‐induced depolarization (AID) of resting membrane potential (RMP) before and after ShK‐223 in the same undifferentiated cell. (e) Scatterplots summarizing RMP and AMP levels before and after ShK‐223 for undifferentiated cells ( n = 14). (f) Voltage‐clamp currents before and after inhibition of Kv1.3 with 100 nM ShK‐223 in an lipopolysaccharides (LPS)‐stimulated microglial cell. (g) Current‐clamp displaying AID of RMP before and after ShK‐223 in the same LPS‐stimulated cell. (h) Scatterplots summarizing RMP and AMP levels for LPS‐treated cells ( n = 8). Statistical significance determined by paired t test. *** p

    Techniques Used: Inhibition

    10) Product Images from "Potassium Channels Kv1.3 and Kir2.1 But Not Kv1.5 Contribute to BV2 Cell Line and Primary Microglial Migration"

    Article Title: Potassium Channels Kv1.3 and Kir2.1 But Not Kv1.5 Contribute to BV2 Cell Line and Primary Microglial Migration

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22042081

    The experimental steps showing the inhibitory effect of potassium channels on microglial migration. ( A ) The monolayer of BV2 microglial cells was scratched with a sterile pipette tip and incubated with the medium or the pharmacological inhibitors or the cells were transfected with small interfering RNA (siRNA); pictures were analyzed at t24h and the inhibition of Kv1.3 and Kir2.1 reduced the rate of migration in the BV2 microglial cell line. ( B ) CX3CR1-eGFP transgenic mice were subjected to SNI or sham surgeries, the ipsilateral spinal cord dorsal horn (SC-DH) was dissected and cultured for 3 h in 8 µm pore inserts, in the culture medium or in the presence of the inhibitors, and the rate of migration was quantified in each condition. Different mechanisms by which the inhibition of the investigated potassium channels may influence the microglial migration rate, are proposed in the outlined box. PBS: phosphate buffer saline; PFA: paraformaldehyde.
    Figure Legend Snippet: The experimental steps showing the inhibitory effect of potassium channels on microglial migration. ( A ) The monolayer of BV2 microglial cells was scratched with a sterile pipette tip and incubated with the medium or the pharmacological inhibitors or the cells were transfected with small interfering RNA (siRNA); pictures were analyzed at t24h and the inhibition of Kv1.3 and Kir2.1 reduced the rate of migration in the BV2 microglial cell line. ( B ) CX3CR1-eGFP transgenic mice were subjected to SNI or sham surgeries, the ipsilateral spinal cord dorsal horn (SC-DH) was dissected and cultured for 3 h in 8 µm pore inserts, in the culture medium or in the presence of the inhibitors, and the rate of migration was quantified in each condition. Different mechanisms by which the inhibition of the investigated potassium channels may influence the microglial migration rate, are proposed in the outlined box. PBS: phosphate buffer saline; PFA: paraformaldehyde.

    Techniques Used: Migration, Transferring, Incubation, Transfection, Small Interfering RNA, Inhibition, Transgenic Assay, Mouse Assay, Cell Culture

    Immunolabeling of potassium channels in cultured BV2 microglial cells. The cells were stained with antibodies targeting Kv1.3 ( A ), Kv1.5 ( B ), Kir2.1 ( C ) and the negative control with Alexa 568 ( D ). The coverslips were mounted using the Prolong antifade with DAPI in blue. The channel was pseudo-colored in green for improved visualization. The images are representative of three independent experiments. Scale bar: 40 µm.
    Figure Legend Snippet: Immunolabeling of potassium channels in cultured BV2 microglial cells. The cells were stained with antibodies targeting Kv1.3 ( A ), Kv1.5 ( B ), Kir2.1 ( C ) and the negative control with Alexa 568 ( D ). The coverslips were mounted using the Prolong antifade with DAPI in blue. The channel was pseudo-colored in green for improved visualization. The images are representative of three independent experiments. Scale bar: 40 µm.

    Techniques Used: Immunolabeling, Cell Culture, Staining, Negative Control

    The contribution of potassium channels in BV2 microglial migration. The representative images show the robust and repetitive scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration in control conditions ( E ) and after the inhibitor at t24h ( F – H ). The histograms show that BV2 microglial cells migrate less after the inhibition of Kv1.3 and Kir2.1 ( B , D ), whereas blocking Kv1.5 has no effect on cellular migration ( C ). Scale bar: 100 µm. All the statistical analysis is represented as Mean ± SD, ***: p
    Figure Legend Snippet: The contribution of potassium channels in BV2 microglial migration. The representative images show the robust and repetitive scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration in control conditions ( E ) and after the inhibitor at t24h ( F – H ). The histograms show that BV2 microglial cells migrate less after the inhibition of Kv1.3 and Kir2.1 ( B , D ), whereas blocking Kv1.5 has no effect on cellular migration ( C ). Scale bar: 100 µm. All the statistical analysis is represented as Mean ± SD, ***: p

    Techniques Used: Migration, Transferring, Inhibition, Blocking Assay

    Electrophysiological properties of BV2 cells. For all Kv1.3, Kv1.5 and Kir2.1 potassium channels, the electrophysiological characteristics are represented by the intensity/voltage (I/V) curves extracted from the step recordings, representing the total current elicited by a voltage protocol starting from −120 mV, in 10 mV increments, to +40 mV ( A – C ), in control conditions and after each specific inhibitor. The inhibition effect of the of each blocker can be seen from the I/V curves. The insets represent the comparison of the current amplitude elicited by a voltage step at −160 mV and +40 mV. All the data are represented as Mean ± SD ( n = 3 independent experiments).
    Figure Legend Snippet: Electrophysiological properties of BV2 cells. For all Kv1.3, Kv1.5 and Kir2.1 potassium channels, the electrophysiological characteristics are represented by the intensity/voltage (I/V) curves extracted from the step recordings, representing the total current elicited by a voltage protocol starting from −120 mV, in 10 mV increments, to +40 mV ( A – C ), in control conditions and after each specific inhibitor. The inhibition effect of the of each blocker can be seen from the I/V curves. The insets represent the comparison of the current amplitude elicited by a voltage step at −160 mV and +40 mV. All the data are represented as Mean ± SD ( n = 3 independent experiments).

    Techniques Used: Inhibition

    BV2 microglial migration after the silencing of the Kv1.3 and Kir2.1 channels. The representative images show the scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration after scrambled and silencing siRNA at t24h ( B – D ) and at t48h ( E – G ). The bar graphs show the BV2 migration after scrambled and silencing siRNA at t24h ( H ) and at t48h ( I ). Scale bar: 100 µm. All the statistical analysis are represented as Mean ± SD, ***: p
    Figure Legend Snippet: BV2 microglial migration after the silencing of the Kv1.3 and Kir2.1 channels. The representative images show the scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration after scrambled and silencing siRNA at t24h ( B – D ) and at t48h ( E – G ). The bar graphs show the BV2 migration after scrambled and silencing siRNA at t24h ( H ) and at t48h ( I ). Scale bar: 100 µm. All the statistical analysis are represented as Mean ± SD, ***: p

    Techniques Used: Migration, Transferring

    The migration of primary microglial cells through inserts with 8 µm pores. ( A ) The migration rate of primary microglial cells after the spared nerve injury (SNI) surgery is reduced compared with sham conditions. Histograms showing the contribution of each potassium channel, Kv1.3, Kv1.5 and Kir2.1, to microglial migration, in both sham ( B – D ) and SNI conditions ( E – G ). All the statistical analysis is represented as Mean ± SD, *: p
    Figure Legend Snippet: The migration of primary microglial cells through inserts with 8 µm pores. ( A ) The migration rate of primary microglial cells after the spared nerve injury (SNI) surgery is reduced compared with sham conditions. Histograms showing the contribution of each potassium channel, Kv1.3, Kv1.5 and Kir2.1, to microglial migration, in both sham ( B – D ) and SNI conditions ( E – G ). All the statistical analysis is represented as Mean ± SD, *: p

    Techniques Used: Migration

    11) Product Images from "Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease"

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI136174

    Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
    Figure Legend Snippet: Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

    Techniques Used: Modification, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Immunohistochemistry, Mouse Assay, Injection, Inhibition, Transfection, Plasmid Preparation, Quantitative RT-PCR, Griess Assay, Luminex

    Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P
    Figure Legend Snippet: Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P

    Techniques Used: Inhibition, Activity Assay, Mouse Assay, High Performance Liquid Chromatography, Western Blot

    Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P
    Figure Legend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P

    Techniques Used: Expressing, Ex Vivo, Derivative Assay, Quantitative RT-PCR, Western Blot, Isolation, Activity Assay

    Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
    Figure Legend Snippet: Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Mouse Assay, Immunohistochemistry, Immunostaining, Staining

    Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
    Figure Legend Snippet: Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Techniques Used: In Silico, Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot, Patch Clamp, Activity Assay, Immunocytochemistry

    Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
    Figure Legend Snippet: Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

    Techniques Used: Cell Culture, Luminex, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Lactate Dehydrogenase Assay, Western Blot, Expressing, Immunocytochemistry

    Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P
    Figure Legend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P

    Techniques Used: Expressing, In Vitro, Patch Clamp, Activity Assay, Quantitative RT-PCR, Western Blot, Immunocytochemistry

    12) Product Images from "Potassium Channels Kv1.3 and Kir2.1 But Not Kv1.5 Contribute to BV2 Cell Line and Primary Microglial Migration"

    Article Title: Potassium Channels Kv1.3 and Kir2.1 But Not Kv1.5 Contribute to BV2 Cell Line and Primary Microglial Migration

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22042081

    The experimental steps showing the inhibitory effect of potassium channels on microglial migration. ( A ) The monolayer of BV2 microglial cells was scratched with a sterile pipette tip and incubated with the medium or the pharmacological inhibitors or the cells were transfected with small interfering RNA (siRNA); pictures were analyzed at t24h and the inhibition of Kv1.3 and Kir2.1 reduced the rate of migration in the BV2 microglial cell line. ( B ) CX3CR1-eGFP transgenic mice were subjected to SNI or sham surgeries, the ipsilateral spinal cord dorsal horn (SC-DH) was dissected and cultured for 3 h in 8 µm pore inserts, in the culture medium or in the presence of the inhibitors, and the rate of migration was quantified in each condition. Different mechanisms by which the inhibition of the investigated potassium channels may influence the microglial migration rate, are proposed in the outlined box. PBS: phosphate buffer saline; PFA: paraformaldehyde.
    Figure Legend Snippet: The experimental steps showing the inhibitory effect of potassium channels on microglial migration. ( A ) The monolayer of BV2 microglial cells was scratched with a sterile pipette tip and incubated with the medium or the pharmacological inhibitors or the cells were transfected with small interfering RNA (siRNA); pictures were analyzed at t24h and the inhibition of Kv1.3 and Kir2.1 reduced the rate of migration in the BV2 microglial cell line. ( B ) CX3CR1-eGFP transgenic mice were subjected to SNI or sham surgeries, the ipsilateral spinal cord dorsal horn (SC-DH) was dissected and cultured for 3 h in 8 µm pore inserts, in the culture medium or in the presence of the inhibitors, and the rate of migration was quantified in each condition. Different mechanisms by which the inhibition of the investigated potassium channels may influence the microglial migration rate, are proposed in the outlined box. PBS: phosphate buffer saline; PFA: paraformaldehyde.

    Techniques Used: Migration, Transferring, Incubation, Transfection, Small Interfering RNA, Inhibition, Transgenic Assay, Mouse Assay, Cell Culture

    Immunolabeling of potassium channels in cultured BV2 microglial cells. The cells were stained with antibodies targeting Kv1.3 ( A ), Kv1.5 ( B ), Kir2.1 ( C ) and the negative control with Alexa 568 ( D ). The coverslips were mounted using the Prolong antifade with DAPI in blue. The channel was pseudo-colored in green for improved visualization. The images are representative of three independent experiments. Scale bar: 40 µm.
    Figure Legend Snippet: Immunolabeling of potassium channels in cultured BV2 microglial cells. The cells were stained with antibodies targeting Kv1.3 ( A ), Kv1.5 ( B ), Kir2.1 ( C ) and the negative control with Alexa 568 ( D ). The coverslips were mounted using the Prolong antifade with DAPI in blue. The channel was pseudo-colored in green for improved visualization. The images are representative of three independent experiments. Scale bar: 40 µm.

    Techniques Used: Immunolabeling, Cell Culture, Staining, Negative Control

    The contribution of potassium channels in BV2 microglial migration. The representative images show the robust and repetitive scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration in control conditions ( E ) and after the inhibitor at t24h ( F – H ). The histograms show that BV2 microglial cells migrate less after the inhibition of Kv1.3 and Kir2.1 ( B , D ), whereas blocking Kv1.5 has no effect on cellular migration ( C ). Scale bar: 100 µm. All the statistical analysis is represented as Mean ± SD, ***: p
    Figure Legend Snippet: The contribution of potassium channels in BV2 microglial migration. The representative images show the robust and repetitive scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration in control conditions ( E ) and after the inhibitor at t24h ( F – H ). The histograms show that BV2 microglial cells migrate less after the inhibition of Kv1.3 and Kir2.1 ( B , D ), whereas blocking Kv1.5 has no effect on cellular migration ( C ). Scale bar: 100 µm. All the statistical analysis is represented as Mean ± SD, ***: p

    Techniques Used: Migration, Transferring, Inhibition, Blocking Assay

    Electrophysiological properties of BV2 cells. For all Kv1.3, Kv1.5 and Kir2.1 potassium channels, the electrophysiological characteristics are represented by the intensity/voltage (I/V) curves extracted from the step recordings, representing the total current elicited by a voltage protocol starting from −120 mV, in 10 mV increments, to +40 mV ( A – C ), in control conditions and after each specific inhibitor. The inhibition effect of the of each blocker can be seen from the I/V curves. The insets represent the comparison of the current amplitude elicited by a voltage step at −160 mV and +40 mV. All the data are represented as Mean ± SD ( n = 3 independent experiments).
    Figure Legend Snippet: Electrophysiological properties of BV2 cells. For all Kv1.3, Kv1.5 and Kir2.1 potassium channels, the electrophysiological characteristics are represented by the intensity/voltage (I/V) curves extracted from the step recordings, representing the total current elicited by a voltage protocol starting from −120 mV, in 10 mV increments, to +40 mV ( A – C ), in control conditions and after each specific inhibitor. The inhibition effect of the of each blocker can be seen from the I/V curves. The insets represent the comparison of the current amplitude elicited by a voltage step at −160 mV and +40 mV. All the data are represented as Mean ± SD ( n = 3 independent experiments).

    Techniques Used: Inhibition

    BV2 microglial migration after the silencing of the Kv1.3 and Kir2.1 channels. The representative images show the scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration after scrambled and silencing siRNA at t24h ( B – D ) and at t48h ( E – G ). The bar graphs show the BV2 migration after scrambled and silencing siRNA at t24h ( H ) and at t48h ( I ). Scale bar: 100 µm. All the statistical analysis are represented as Mean ± SD, ***: p
    Figure Legend Snippet: BV2 microglial migration after the silencing of the Kv1.3 and Kir2.1 channels. The representative images show the scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration after scrambled and silencing siRNA at t24h ( B – D ) and at t48h ( E – G ). The bar graphs show the BV2 migration after scrambled and silencing siRNA at t24h ( H ) and at t48h ( I ). Scale bar: 100 µm. All the statistical analysis are represented as Mean ± SD, ***: p

    Techniques Used: Migration, Transferring

    The migration of primary microglial cells through inserts with 8 µm pores. ( A ) The migration rate of primary microglial cells after the spared nerve injury (SNI) surgery is reduced compared with sham conditions. Histograms showing the contribution of each potassium channel, Kv1.3, Kv1.5 and Kir2.1, to microglial migration, in both sham ( B – D ) and SNI conditions ( E – G ). All the statistical analysis is represented as Mean ± SD, *: p
    Figure Legend Snippet: The migration of primary microglial cells through inserts with 8 µm pores. ( A ) The migration rate of primary microglial cells after the spared nerve injury (SNI) surgery is reduced compared with sham conditions. Histograms showing the contribution of each potassium channel, Kv1.3, Kv1.5 and Kir2.1, to microglial migration, in both sham ( B – D ) and SNI conditions ( E – G ). All the statistical analysis is represented as Mean ± SD, *: p

    Techniques Used: Migration

    13) Product Images from "Comparison of cellular effects of starch-coated SPIONs and poly(lactic-co-glycolic acid) matrix nanoparticles on human monocytes"

    Article Title: Comparison of cellular effects of starch-coated SPIONs and poly(lactic-co-glycolic acid) matrix nanoparticles on human monocytes

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S106540

    Validation of voltage-gated potassium channels. Notes: Fluorescent staining of the voltage-gated potassium channel proteins ( A and B ) Kv1.3 and ( C and D ) Kv7.1 in ( A , C , and E ) primary monocytes or ( B , D , and F ) the MM6 cell line with an ( E and F ) unspecific IgG control. Cells were stained with either rabbit anti-Kv1.3 (3 µg/mL; APC-002; Alomone Labs) or rabbit anti-Kv7.1 (4 µg/mL; AB5932; Millipore) or unspecific rabbit IgG (4 µg/mL; ab27478; Abcam) and goat anti-rabbit IgG (10 µg/mL; A11012; Thermo Fisher Scientific) coupled to Alexa Fluor 594. Abbreviation: IgG, immunoglobulin G.
    Figure Legend Snippet: Validation of voltage-gated potassium channels. Notes: Fluorescent staining of the voltage-gated potassium channel proteins ( A and B ) Kv1.3 and ( C and D ) Kv7.1 in ( A , C , and E ) primary monocytes or ( B , D , and F ) the MM6 cell line with an ( E and F ) unspecific IgG control. Cells were stained with either rabbit anti-Kv1.3 (3 µg/mL; APC-002; Alomone Labs) or rabbit anti-Kv7.1 (4 µg/mL; AB5932; Millipore) or unspecific rabbit IgG (4 µg/mL; ab27478; Abcam) and goat anti-rabbit IgG (10 µg/mL; A11012; Thermo Fisher Scientific) coupled to Alexa Fluor 594. Abbreviation: IgG, immunoglobulin G.

    Techniques Used: Staining

    Demonstration of voltage-gated potassium channels in primary cells and the MM6 cells. Notes: Western blot of voltage-gated potassium channels ( A ) Kv1.3 and ( B ) Kv7.1 in the leukemic monocyte cell line MM6 and primary monocytes (M0). Primary antibodies used were rabbit anti-Kv1.3 (3 µg/mL; APC-002; Alomone Labs) or rabbit anti-Kv7.1 (4 µg/mL; AB5932; Millipore, Billerica, MA, USA) and a goat anti-rabbit IgG coupled to alkaline phosphatase (50 ng/mL; Abcam). Bound antibody was stained by the NBT/BCIP technique (Sigma-Aldrich Co.). Abbreviations: IgG, immunoglobulin G; NBT/BCIP, nitro-blue tetrazolium chloride/5-bromo-4-chloro-3′-indoylphosphate p -toluidine salt.
    Figure Legend Snippet: Demonstration of voltage-gated potassium channels in primary cells and the MM6 cells. Notes: Western blot of voltage-gated potassium channels ( A ) Kv1.3 and ( B ) Kv7.1 in the leukemic monocyte cell line MM6 and primary monocytes (M0). Primary antibodies used were rabbit anti-Kv1.3 (3 µg/mL; APC-002; Alomone Labs) or rabbit anti-Kv7.1 (4 µg/mL; AB5932; Millipore, Billerica, MA, USA) and a goat anti-rabbit IgG coupled to alkaline phosphatase (50 ng/mL; Abcam). Bound antibody was stained by the NBT/BCIP technique (Sigma-Aldrich Co.). Abbreviations: IgG, immunoglobulin G; NBT/BCIP, nitro-blue tetrazolium chloride/5-bromo-4-chloro-3′-indoylphosphate p -toluidine salt.

    Techniques Used: Western Blot, Staining

    14) Product Images from "Activated T cells Inhibit Neurogenesis by Releasing Granzyme B: Rescue by Kv1.3 blockers"

    Article Title: Activated T cells Inhibit Neurogenesis by Releasing Granzyme B: Rescue by Kv1.3 blockers

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

    doi: 10.1523/JNEUROSCI.0311-10.2010

    MgTX protects NPC against GrB-induced effects in rat dentate gyrus Eight weeks old female rats were stereotaxically injected in the dentate gyrus (DG) with GrB (1 ug), MgTX (10 ng) +GrB (1 ug) or vehicle control (PBS). After seven days, the rats received BrdU (100 mg/kg, i.p.) two hours before being euthanized. Serial sections of the hippocampus and DG were analyzed by quantitative immunohistochemistry. Sections were immunostained with anti-BrdU (red), and anti-NeuN (green) or anti-Kv1.3 (green). DAPI (blue) was used for nuclear staining. BrdU positive cells in the subgranular zone (SGZ) of every sixth section were counted in a blinded fashion, and normalized to the volume of each granule cell layer (GCL). (A) Representative immunostained sections are shown from each of the treated groups. (B) Quantitative analysis shows decreased numbers of BrdU staining cells with GrB and restoration with MgTX (C) Kv1.3 (green) fluorescence immunohistochemistry shows GrB increases Kv1.3 expression in the dentate gyrus compared to vehicle control. The cellular localization of Kv1.3 (green) was characterized by co-localization studies with BrdU (red).
    Figure Legend Snippet: MgTX protects NPC against GrB-induced effects in rat dentate gyrus Eight weeks old female rats were stereotaxically injected in the dentate gyrus (DG) with GrB (1 ug), MgTX (10 ng) +GrB (1 ug) or vehicle control (PBS). After seven days, the rats received BrdU (100 mg/kg, i.p.) two hours before being euthanized. Serial sections of the hippocampus and DG were analyzed by quantitative immunohistochemistry. Sections were immunostained with anti-BrdU (red), and anti-NeuN (green) or anti-Kv1.3 (green). DAPI (blue) was used for nuclear staining. BrdU positive cells in the subgranular zone (SGZ) of every sixth section were counted in a blinded fashion, and normalized to the volume of each granule cell layer (GCL). (A) Representative immunostained sections are shown from each of the treated groups. (B) Quantitative analysis shows decreased numbers of BrdU staining cells with GrB and restoration with MgTX (C) Kv1.3 (green) fluorescence immunohistochemistry shows GrB increases Kv1.3 expression in the dentate gyrus compared to vehicle control. The cellular localization of Kv1.3 (green) was characterized by co-localization studies with BrdU (red).

    Techniques Used: Injection, Immunohistochemistry, Staining, BrdU Staining, Fluorescence, Expressing

    Kv1.3 expression in GrB-treated NPC (A) Kv1.3 gene expression was detected using real-time PCR in NPC cultures treated with GrB (4 nM) for 1-3 hours. GADPH was used as an internal control. Dose-dependent increase in Kv1.3 mRNA expression was noted. Data represent mean ± SEM from four independent experiments. (B) NPC cultures were treated with GrB (4nM) for 24 hours and immunostained with antibodies to Kv1.3 and nestin (Red: nestin, Green: Kv1.3). (C) NPC cultures were treated with GrB (1-4 nM) for 24 hours and the cell lysates were collected for detecting Kv1.3 production using western-blot analysis. This shows a dose-dependent increase in Kv1.3 detection.
    Figure Legend Snippet: Kv1.3 expression in GrB-treated NPC (A) Kv1.3 gene expression was detected using real-time PCR in NPC cultures treated with GrB (4 nM) for 1-3 hours. GADPH was used as an internal control. Dose-dependent increase in Kv1.3 mRNA expression was noted. Data represent mean ± SEM from four independent experiments. (B) NPC cultures were treated with GrB (4nM) for 24 hours and immunostained with antibodies to Kv1.3 and nestin (Red: nestin, Green: Kv1.3). (C) NPC cultures were treated with GrB (1-4 nM) for 24 hours and the cell lysates were collected for detecting Kv1.3 production using western-blot analysis. This shows a dose-dependent increase in Kv1.3 detection.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Kv1.3 mediates effect of GrB on NPC neurogenesis To monitor NPC neurogenesis NPC cultures were cultured in differentiating media for 4-7 days. Cells were immunostained with anti-beta-III tubulin antibody (red) and anti-GFAP antiserum (green). Beta-III tubulin positive neurons were counted in each well and the cell numbers were expressed as percentage of total cells. Cells in 5 pre-assigned fields (approx 200 cells/field) were counted on each cover slip and three cover slips were counted in every group. Data represents mean ± SEM from three experiments. (A) Kv1.3 selective blocker MgTX (10 nM) prevented the effects of GrB (4 nM) on NPC neurogenesis. (B) MgTX also attenuated the effect of supernatants from activated T cells (Ac-T) on NPC neurogenesis. (C) Transfection of Kv1.3 siRNA (25 nM final concentration) but not nonspecific control (NSi) into NPC blocked the effects of GrB (4 nM) on NPC neurogenesis. (D) Representative photomicrographs immunostaining for beta-III tubulin.
    Figure Legend Snippet: Kv1.3 mediates effect of GrB on NPC neurogenesis To monitor NPC neurogenesis NPC cultures were cultured in differentiating media for 4-7 days. Cells were immunostained with anti-beta-III tubulin antibody (red) and anti-GFAP antiserum (green). Beta-III tubulin positive neurons were counted in each well and the cell numbers were expressed as percentage of total cells. Cells in 5 pre-assigned fields (approx 200 cells/field) were counted on each cover slip and three cover slips were counted in every group. Data represents mean ± SEM from three experiments. (A) Kv1.3 selective blocker MgTX (10 nM) prevented the effects of GrB (4 nM) on NPC neurogenesis. (B) MgTX also attenuated the effect of supernatants from activated T cells (Ac-T) on NPC neurogenesis. (C) Transfection of Kv1.3 siRNA (25 nM final concentration) but not nonspecific control (NSi) into NPC blocked the effects of GrB (4 nM) on NPC neurogenesis. (D) Representative photomicrographs immunostaining for beta-III tubulin.

    Techniques Used: Cell Culture, Transfection, Concentration Assay, Immunostaining

    PTX attenuated GrB-induced Kv1.3 expression NPC cultures were pretreated with PTX for 1 h prior to GrB treatment. The cells were lysed 3 h after GrB treatment. Kv1.3 mRNA was detected using real-time PCR. The results were expressed as folds compared to control, Data were from three independent experiments.
    Figure Legend Snippet: PTX attenuated GrB-induced Kv1.3 expression NPC cultures were pretreated with PTX for 1 h prior to GrB treatment. The cells were lysed 3 h after GrB treatment. Kv1.3 mRNA was detected using real-time PCR. The results were expressed as folds compared to control, Data were from three independent experiments.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    15) Product Images from "Localization of Kv1.3 channels in presynaptic terminals of brainstem auditory neurons"

    Article Title: Localization of Kv1.3 channels in presynaptic terminals of brainstem auditory neurons

    Journal: The Journal of comparative neurology

    doi: 10.1002/cne.22393

    Comparison of localization patterns of Kv1.3 in MNTB with Kv1.1, Kv1.2 and Kv1.6. Top row (A–C) shows double immunofluorescence labeling of Kv1.3 with Kv1.1 (A), A and B show Kv1.3 and Kv1.1 immunolabeling and C shows the merged fluorescence images.
    Figure Legend Snippet: Comparison of localization patterns of Kv1.3 in MNTB with Kv1.1, Kv1.2 and Kv1.6. Top row (A–C) shows double immunofluorescence labeling of Kv1.3 with Kv1.1 (A), A and B show Kv1.3 and Kv1.1 immunolabeling and C shows the merged fluorescence images.

    Techniques Used: Immunofluorescence, Labeling, Immunolabeling, Fluorescence

    Gradients of Kv1.3 immunoreactivity in sagittal and coronal sections of the MNTB. A. Sagittal section of MNTB immunostained for Kv 1.3. (scale bar, 20 μm). B and C. Coronal (B) and sagittal (C) sections were divided into 5 equal zones for quantification
    Figure Legend Snippet: Gradients of Kv1.3 immunoreactivity in sagittal and coronal sections of the MNTB. A. Sagittal section of MNTB immunostained for Kv 1.3. (scale bar, 20 μm). B and C. Coronal (B) and sagittal (C) sections were divided into 5 equal zones for quantification

    Techniques Used:

    Immunostaining for the Kv1.3 potassium channel subunit in the MNTB
    Figure Legend Snippet: Immunostaining for the Kv1.3 potassium channel subunit in the MNTB

    Techniques Used: Immunostaining

    Electron microscopic immunogold labeling of Kv 1.3 in the MNTB of Wild-type and Kv 1.3 −/− mice. A and B. Examples of sections stained for Kv1.3 using 10 nm gold particles. Particles are localized primarily to pre-synaptic structures,
    Figure Legend Snippet: Electron microscopic immunogold labeling of Kv 1.3 in the MNTB of Wild-type and Kv 1.3 −/− mice. A and B. Examples of sections stained for Kv1.3 using 10 nm gold particles. Particles are localized primarily to pre-synaptic structures,

    Techniques Used: Labeling, Mouse Assay, Staining

    Co-localization of Kv 1.3 with syntaxin, synaptophysin and synaptotagmin. Double immunofluorescence labeling of Kv1.3 with syntaxin (top panels A – E ), synaptophysin ( center panels F – J ) and synaptotagmin ( bottom panels K – O ). Kv 1.3
    Figure Legend Snippet: Co-localization of Kv 1.3 with syntaxin, synaptophysin and synaptotagmin. Double immunofluorescence labeling of Kv1.3 with syntaxin (top panels A – E ), synaptophysin ( center panels F – J ) and synaptotagmin ( bottom panels K – O ). Kv 1.3

    Techniques Used: Immunofluorescence, Labeling

    Electron microscopic co-localization of Kv1.3 with synaptophysin in wild-type and Kv1.3−/− animals. Double immunogold labeling was carried out using large (10 nm) gold particles to detect synaptophysin and smaller (5nm) particles to localize
    Figure Legend Snippet: Electron microscopic co-localization of Kv1.3 with synaptophysin in wild-type and Kv1.3−/− animals. Double immunogold labeling was carried out using large (10 nm) gold particles to detect synaptophysin and smaller (5nm) particles to localize

    Techniques Used: Labeling

    Immunolocalization of Kv1.3 potassium channels subunits in the MNTB using diaminobenzidine labeling. A. Low power view of part of a coronal section of brainstem, showing bilateral immunostaining of MNTBs in a wild-type (wt) mouse (white arrows). B. Image
    Figure Legend Snippet: Immunolocalization of Kv1.3 potassium channels subunits in the MNTB using diaminobenzidine labeling. A. Low power view of part of a coronal section of brainstem, showing bilateral immunostaining of MNTBs in a wild-type (wt) mouse (white arrows). B. Image

    Techniques Used: Labeling, Immunostaining

    16) Product Images from "O2-sensitive K+ channels: role of the Kv1.2 ?-subunit in mediating the hypoxic response"

    Article Title: O2-sensitive K+ channels: role of the Kv1.2 ?-subunit in mediating the hypoxic response

    Journal: The Journal of Physiology

    doi: 10.1111/j.1469-7793.2000.00783.x

    Expression of the Kv1.2 and Kv2.1 α-subunits of K + channels in PC12 cells and specificity of anti-Kv1.2 antibody A , Kv1.2 and Kv2.1 polypeptide expression in PC12 cell total lysate. Immunoblots of rat PC12 cell protein (40 μg) were incubated with affinity-purified anti-Kv1.2 or anti-Kv2.1 antibodies. Molecular mass markers are indicated on the left in kilodaltons (kDa). B , immunoblots of GST-fusion proteins (50 ng) for Kv1.3 and Kv1.2 (indicated at the bottom of the blot) were incubated with anti-Kv1.2 antibody (left panel) or anti-Kv1.3 antibody (right panel). C , immunostaining of PC12 cells with anti-Kv1.2 antibody. Panel a , background staining of PC12 cells that were subjected to all steps in the staining protocol, except that the primary antibody was omitted. Panel b , labelling of PC12 cell membranes with anti-Kv1.2 antibody. Panel c , immunostaining of PC12 cell with anti-Kv1.2 antibody pre-incubated with the antigen against which the antibody is directed. The intensity of the fluorescent signal is comparable to the background fluorescence observed in panel a . Scale bar = 20 μm and applies to all panels in C .
    Figure Legend Snippet: Expression of the Kv1.2 and Kv2.1 α-subunits of K + channels in PC12 cells and specificity of anti-Kv1.2 antibody A , Kv1.2 and Kv2.1 polypeptide expression in PC12 cell total lysate. Immunoblots of rat PC12 cell protein (40 μg) were incubated with affinity-purified anti-Kv1.2 or anti-Kv2.1 antibodies. Molecular mass markers are indicated on the left in kilodaltons (kDa). B , immunoblots of GST-fusion proteins (50 ng) for Kv1.3 and Kv1.2 (indicated at the bottom of the blot) were incubated with anti-Kv1.2 antibody (left panel) or anti-Kv1.3 antibody (right panel). C , immunostaining of PC12 cells with anti-Kv1.2 antibody. Panel a , background staining of PC12 cells that were subjected to all steps in the staining protocol, except that the primary antibody was omitted. Panel b , labelling of PC12 cell membranes with anti-Kv1.2 antibody. Panel c , immunostaining of PC12 cell with anti-Kv1.2 antibody pre-incubated with the antigen against which the antibody is directed. The intensity of the fluorescent signal is comparable to the background fluorescence observed in panel a . Scale bar = 20 μm and applies to all panels in C .

    Techniques Used: Expressing, Western Blot, Incubation, Affinity Purification, Immunostaining, Staining, Fluorescence

    17) Product Images from "Nuclear Localization and Functional Characteristics of Voltage-gated Potassium Channel Kv1.3 *"

    Article Title: Nuclear Localization and Functional Characteristics of Voltage-gated Potassium Channel Kv1.3 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.561324

    Subcellular localization of Kv1.3 protein in cancer cells and human brain tissues. Western blot ( WB ) analysis of Kv1.3 shows labeling within the nuclear fraction of three cancer cell lines (MCF7, A549, and SNU-484) ( n = 4) ( A ) and human brain tissue extracts
    Figure Legend Snippet: Subcellular localization of Kv1.3 protein in cancer cells and human brain tissues. Western blot ( WB ) analysis of Kv1.3 shows labeling within the nuclear fraction of three cancer cell lines (MCF7, A549, and SNU-484) ( n = 4) ( A ) and human brain tissue extracts

    Techniques Used: Western Blot, Labeling

    Up-regulation of phosphorylated CREB and c-Fos induced by Kv1.3 blockers. Representative gel images of Western blots ( WB ) and the relative expression level of pCREB ( n = 4) and CREB ( n = 4) induced by 1 n m MgTX in the isolated nuclei of A549 ( A ), and
    Figure Legend Snippet: Up-regulation of phosphorylated CREB and c-Fos induced by Kv1.3 blockers. Representative gel images of Western blots ( WB ) and the relative expression level of pCREB ( n = 4) and CREB ( n = 4) induced by 1 n m MgTX in the isolated nuclei of A549 ( A ), and

    Techniques Used: Western Blot, Expressing, Isolation

    Co-assembly of UBF1 and nuclear Kv1.3 channel. A , MS/MS spectra of the UBF1-specific peptide obtained by mass spectrometry. The b ions originate from the cleavage of the peptide backbone with N-terminal charge retention, and the y ions indicate peptide
    Figure Legend Snippet: Co-assembly of UBF1 and nuclear Kv1.3 channel. A , MS/MS spectra of the UBF1-specific peptide obtained by mass spectrometry. The b ions originate from the cleavage of the peptide backbone with N-terminal charge retention, and the y ions indicate peptide

    Techniques Used: Mass Spectrometry

    Subcellular localization of Kv1.3 protein in human Jurkat cells. After subcellular fractionation, a band representing Kv1.3 is shown. The expression of Kv1.3 was detected in the plasma and mitochondria membrane and nuclear fractions. The specificity of
    Figure Legend Snippet: Subcellular localization of Kv1.3 protein in human Jurkat cells. After subcellular fractionation, a band representing Kv1.3 is shown. The expression of Kv1.3 was detected in the plasma and mitochondria membrane and nuclear fractions. The specificity of

    Techniques Used: Fractionation, Expressing

    Sp1 transcription factor interaction with the Kv1.3 promoter and down-regulated Kv1.3 protein expression by inhibition of Sp1. A , a ChIP assay showing Sp1 binding to the Kv1.3 promoter in A549 cells ( n = 3). Input DNA was used as a control, and rabbit
    Figure Legend Snippet: Sp1 transcription factor interaction with the Kv1.3 promoter and down-regulated Kv1.3 protein expression by inhibition of Sp1. A , a ChIP assay showing Sp1 binding to the Kv1.3 promoter in A549 cells ( n = 3). Input DNA was used as a control, and rabbit

    Techniques Used: Expressing, Inhibition, Chromatin Immunoprecipitation, Binding Assay

    The effect of MgTX on nuclear membrane potential of the isolated nuclei from A549 cells. A , Western blot ( WB ) analysis indicated that Kv1.3 exists in the nuclear membranes of A549 cells. Nuclear membrane fractionation was confirmed using Sp1 (a nucleoplasmic
    Figure Legend Snippet: The effect of MgTX on nuclear membrane potential of the isolated nuclei from A549 cells. A , Western blot ( WB ) analysis indicated that Kv1.3 exists in the nuclear membranes of A549 cells. Nuclear membrane fractionation was confirmed using Sp1 (a nucleoplasmic

    Techniques Used: Isolation, Western Blot, Fractionation

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    Alomone Labs rabbit polyclonal anti kv1 3 antibody
    <t>Kv1.3</t> channels are recruited at the interface between CD3/CD28 beads and T cells
    Rabbit Polyclonal Anti Kv1 3 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti kv1 3 antibody/product/Alomone Labs
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    Alomone Labs kv1 3
    Callosal <t>Kv1.3</t> channel protein in axons and glia is altered after injury and with CFZ treatment A. Confocal overlay showing Kv1.3 ( red ) and Kv1.2 ( green ) in rat corpus callosum 24h following midline fluid percussion TBI. Low magnification shows that each channel protein is found in reactive glia around callosal vessels (arrowheads) and along axon bundles (arrows). Inset shows paired paranodal distribution of Kv1.3 and Kv1.2 channels, some nodes with co-localization (yellow arrow), others with single channel expression (green, red arrows). B . Confocal overlays showing Kv1.3 in callosal astrocytes of sham injured (GFAP+, left-arrows; inset shows cell body and perivascular co-localization) and microglia of 24h postinjury cases (IBA1+, right-arrows). C. Western blot (WB) of protein extracts from 24h postinjury corpus callosum revealed that TBI reduced 67kD Kv1.3 levels and that CFZ treatment reversed loss of Kv1.3 expression. Data expressed as percent of paired untreated sham controls run on same blot. Lanes representative of group effects are shown in each panel. (ANOVA, *p
    Kv1 3, 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
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    Kv1.3 channels are recruited at the interface between CD3/CD28 beads and T cells

    Journal:

    Article Title: ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1 , 2

    doi:

    Figure Lengend Snippet: Kv1.3 channels are recruited at the interface between CD3/CD28 beads and T cells

    Article Snippet: The primary antibodies used for detecting Kv1.3 proteins were either a rabbit polyclonal anti-Kv1.3 antibody against an epitope on the C-terminal domain of the protein (Alomone, Jerusalem, Israel) or an extracellular epitope (Sigma-Aldrich).

    Techniques:

    Kv1.3 channels in T lymphocytes from patients with SLE display biophysical and pharmacological properties similar to those in healthy T cells

    Journal:

    Article Title: ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1 , 2

    doi:

    Figure Lengend Snippet: Kv1.3 channels in T lymphocytes from patients with SLE display biophysical and pharmacological properties similar to those in healthy T cells

    Article Snippet: The primary antibodies used for detecting Kv1.3 proteins were either a rabbit polyclonal anti-Kv1.3 antibody against an epitope on the C-terminal domain of the protein (Alomone, Jerusalem, Israel) or an extracellular epitope (Sigma-Aldrich).

    Techniques:

    Comparison of the rates of Kv1.3 channel compartmentalization in the IS in normal and SLE T cells

    Journal:

    Article Title: ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1 , 2

    doi:

    Figure Lengend Snippet: Comparison of the rates of Kv1.3 channel compartmentalization in the IS in normal and SLE T cells

    Article Snippet: The primary antibodies used for detecting Kv1.3 proteins were either a rabbit polyclonal anti-Kv1.3 antibody against an epitope on the C-terminal domain of the protein (Alomone, Jerusalem, Israel) or an extracellular epitope (Sigma-Aldrich).

    Techniques:

    Electrophysiological and pharmacological properties of Kv1.3 channels in SLE T cells

    Journal:

    Article Title: ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1 , 2

    doi:

    Figure Lengend Snippet: Electrophysiological and pharmacological properties of Kv1.3 channels in SLE T cells

    Article Snippet: The primary antibodies used for detecting Kv1.3 proteins were either a rabbit polyclonal anti-Kv1.3 antibody against an epitope on the C-terminal domain of the protein (Alomone, Jerusalem, Israel) or an extracellular epitope (Sigma-Aldrich).

    Techniques:

    APC-T cell activation induces differential reorganization of Kv1.3 channels in the IS formed with resting healthy and SLE T cells

    Journal:

    Article Title: ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1 , 2

    doi:

    Figure Lengend Snippet: APC-T cell activation induces differential reorganization of Kv1.3 channels in the IS formed with resting healthy and SLE T cells

    Article Snippet: The primary antibodies used for detecting Kv1.3 proteins were either a rabbit polyclonal anti-Kv1.3 antibody against an epitope on the C-terminal domain of the protein (Alomone, Jerusalem, Israel) or an extracellular epitope (Sigma-Aldrich).

    Techniques: Activation Assay

    Differential kinetics of Kv1.3 channel reorganization in the IS

    Journal:

    Article Title: ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1 , 2

    doi:

    Figure Lengend Snippet: Differential kinetics of Kv1.3 channel reorganization in the IS

    Article Snippet: The primary antibodies used for detecting Kv1.3 proteins were either a rabbit polyclonal anti-Kv1.3 antibody against an epitope on the C-terminal domain of the protein (Alomone, Jerusalem, Israel) or an extracellular epitope (Sigma-Aldrich).

    Techniques:

    Kv1.3 channel recruitment in the IS in activated healthy T cells parallels SLE T lymphocytes

    Journal:

    Article Title: ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1 , 2

    doi:

    Figure Lengend Snippet: Kv1.3 channel recruitment in the IS in activated healthy T cells parallels SLE T lymphocytes

    Article Snippet: The primary antibodies used for detecting Kv1.3 proteins were either a rabbit polyclonal anti-Kv1.3 antibody against an epitope on the C-terminal domain of the protein (Alomone, Jerusalem, Israel) or an extracellular epitope (Sigma-Aldrich).

    Techniques:

    The kinetics of Kv1.3 redistribution in the immunological synapse of SLE T cells resemble those of pre-activated normal T cells

    Journal:

    Article Title: ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1ALTERED DYNAMICS OF Kv1.3 CHANNEL COMPARTMENTALIZATION IN THE IMMUNOLOGICAL SYNAPSE IN SYSTEMIC LUPUS ERYTHEMATOSUS 1 , 2

    doi:

    Figure Lengend Snippet: The kinetics of Kv1.3 redistribution in the immunological synapse of SLE T cells resemble those of pre-activated normal T cells

    Article Snippet: The primary antibodies used for detecting Kv1.3 proteins were either a rabbit polyclonal anti-Kv1.3 antibody against an epitope on the C-terminal domain of the protein (Alomone, Jerusalem, Israel) or an extracellular epitope (Sigma-Aldrich).

    Techniques:

    Expression of the potassium channel Kv1.3. Protein extracts were prepared from control and iron-fed C8B4 microglia. Western blot analysis was carried out to determine the level of Kv1.3 in the microglia. Bands for the protein were observed in both control and iron-fed microglia. Levels of tubulin were also determined to verify protein loading. The results showed a significant ( p

    Journal: Biomolecules

    Article Title: Model Senescent Microglia Induce Disease Related Changes in α-Synuclein Expression and Activity

    doi: 10.3390/biom8030067

    Figure Lengend Snippet: Expression of the potassium channel Kv1.3. Protein extracts were prepared from control and iron-fed C8B4 microglia. Western blot analysis was carried out to determine the level of Kv1.3 in the microglia. Bands for the protein were observed in both control and iron-fed microglia. Levels of tubulin were also determined to verify protein loading. The results showed a significant ( p

    Article Snippet: Anti-l-ferritin mouse monoclonal (SC-25616, Santa Cruz, Dallas, TX, USA) was used at 1:5000, anti-Kv1.3 rabbit polyclonal was used at 1:400 (APC101, Alomone, Jerusalem, Israel), and anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mouse monoclonal was used at 1:2000 (6C5, Abcam).

    Techniques: Expressing, Western Blot

    Recognition of Kv1.3 protein by α-AU13 antiserum by Western blot analysis Cell lysates from Kv1.3 transfected HEK293 cells, separated by SDS-PAGE and visualized by Western blot analysis with various dilutions of the antiserum from 1 : 500 to 1:10 000.

    Journal: The Journal of Physiology

    Article Title: Neurotrophin modulation of voltage-gated potassium channels in rat through TrkB receptors is time and sensory experience dependent

    doi: 10.1113/jphysiol.2002.017376

    Figure Lengend Snippet: Recognition of Kv1.3 protein by α-AU13 antiserum by Western blot analysis Cell lysates from Kv1.3 transfected HEK293 cells, separated by SDS-PAGE and visualized by Western blot analysis with various dilutions of the antiserum from 1 : 500 to 1:10 000.

    Article Snippet: Second, the antiserum generated against Kv1.3 (α-AU13; see Methods), as well as several other anti-Kv1.3 antisera that were either internally generated (α-AU11 and α-AU12) or commercially available (Alomone Laboratories), were tested for the ability to recognize cloned Kv1.3 protein as transiently expressed in HEK293 cells and visualized in Western blots. α-AU13 specifically recognized Kv1.3 at serial dilutions of the antiserum from 1 : 500 to 1 : 10 000 ( ) and was therefore the antibody used in all subsequent experiments. α-AU13 was further characterized in native olfactory bulb membranes, where the appropriate molecular weight band was preabsorbed by incubation with the 46 amino acid peptide used to generate the antiserum (data not shown).

    Techniques: Western Blot, Transfection, SDS Page

    Sensory deprivation by unilateral naris occlusion alters BDNF-stimulated tyrosine phosphorylation of Kv1.3 channel P1 animals were left naris occluded by cauterization and raised with odour sensory deprivation to this naris from P20 to P25. A , olfactory bulbs contralateral (non-occluded) and ipsilateral (occluded) to the cauterized naris were then harvested, stimulated with BDNF and immunoprecipitated with anti-Kv1.3 and blotted with anti-4G10. Total tyrosine phosphorylation of Kv1.3 is indicated by the arrow. B , histogram of the mean increase in tyrosine phosphorylation of Kv1.3 by acute BDNF stimulation comparing non-occluded versus sensory-deprived conditions (occluded). Pixel values were calculated by quantitative densitometry. The difference in pixel density between unstimulated and BDNF-stimulated olfactory bulbs was plotted for occluded and non-occluded naris conditions, where 0 = no change in Kv1.3 phosphorylation with BDNF treatment. * Significantly different by Student's paired t test, P

    Journal: The Journal of Physiology

    Article Title: Neurotrophin modulation of voltage-gated potassium channels in rat through TrkB receptors is time and sensory experience dependent

    doi: 10.1113/jphysiol.2002.017376

    Figure Lengend Snippet: Sensory deprivation by unilateral naris occlusion alters BDNF-stimulated tyrosine phosphorylation of Kv1.3 channel P1 animals were left naris occluded by cauterization and raised with odour sensory deprivation to this naris from P20 to P25. A , olfactory bulbs contralateral (non-occluded) and ipsilateral (occluded) to the cauterized naris were then harvested, stimulated with BDNF and immunoprecipitated with anti-Kv1.3 and blotted with anti-4G10. Total tyrosine phosphorylation of Kv1.3 is indicated by the arrow. B , histogram of the mean increase in tyrosine phosphorylation of Kv1.3 by acute BDNF stimulation comparing non-occluded versus sensory-deprived conditions (occluded). Pixel values were calculated by quantitative densitometry. The difference in pixel density between unstimulated and BDNF-stimulated olfactory bulbs was plotted for occluded and non-occluded naris conditions, where 0 = no change in Kv1.3 phosphorylation with BDNF treatment. * Significantly different by Student's paired t test, P

    Article Snippet: Second, the antiserum generated against Kv1.3 (α-AU13; see Methods), as well as several other anti-Kv1.3 antisera that were either internally generated (α-AU11 and α-AU12) or commercially available (Alomone Laboratories), were tested for the ability to recognize cloned Kv1.3 protein as transiently expressed in HEK293 cells and visualized in Western blots. α-AU13 specifically recognized Kv1.3 at serial dilutions of the antiserum from 1 : 500 to 1 : 10 000 ( ) and was therefore the antibody used in all subsequent experiments. α-AU13 was further characterized in native olfactory bulb membranes, where the appropriate molecular weight band was preabsorbed by incubation with the 46 amino acid peptide used to generate the antiserum (data not shown).

    Techniques: Immunoprecipitation

    Acute BDNF stimulation increases the tyrosine phosphorylation of Kv1.3 in the rat olfactory bulb A , olfactory bulbs were stimulated with or without 100 ng ml −1 of BDNF in PBS for 20 min, homogenized, immunoprecipitated with anti-phosphotyrosine antiserum (anti-4G10), separated by SDS-PAGE, and Western blots were probed with anti-Kv1.3 (α-AU13). Total tyrosine phosphorylation of Kv1.3 is shown in the bottom panel. The heavy chain of IgG is also indicated below that of Kv1.3. Ten micrograms of cell lysate were blotted for α-AU13 and α-TrkB, respectively, to confirm equivalent protein expression of the channel and receptor under BDNF-stimulated and unstimulated conditions (top panels). B , histogram of quantitative densitometry of four experiments as in A . Mean pixel density of Kv1.3 tyrosine phosphorylation under control versus BDNF-stimulated conditions. * Significantly different, Arc Sin transformation for percentile data, Student's t test P

    Journal: The Journal of Physiology

    Article Title: Neurotrophin modulation of voltage-gated potassium channels in rat through TrkB receptors is time and sensory experience dependent

    doi: 10.1113/jphysiol.2002.017376

    Figure Lengend Snippet: Acute BDNF stimulation increases the tyrosine phosphorylation of Kv1.3 in the rat olfactory bulb A , olfactory bulbs were stimulated with or without 100 ng ml −1 of BDNF in PBS for 20 min, homogenized, immunoprecipitated with anti-phosphotyrosine antiserum (anti-4G10), separated by SDS-PAGE, and Western blots were probed with anti-Kv1.3 (α-AU13). Total tyrosine phosphorylation of Kv1.3 is shown in the bottom panel. The heavy chain of IgG is also indicated below that of Kv1.3. Ten micrograms of cell lysate were blotted for α-AU13 and α-TrkB, respectively, to confirm equivalent protein expression of the channel and receptor under BDNF-stimulated and unstimulated conditions (top panels). B , histogram of quantitative densitometry of four experiments as in A . Mean pixel density of Kv1.3 tyrosine phosphorylation under control versus BDNF-stimulated conditions. * Significantly different, Arc Sin transformation for percentile data, Student's t test P

    Article Snippet: Second, the antiserum generated against Kv1.3 (α-AU13; see Methods), as well as several other anti-Kv1.3 antisera that were either internally generated (α-AU11 and α-AU12) or commercially available (Alomone Laboratories), were tested for the ability to recognize cloned Kv1.3 protein as transiently expressed in HEK293 cells and visualized in Western blots. α-AU13 specifically recognized Kv1.3 at serial dilutions of the antiserum from 1 : 500 to 1 : 10 000 ( ) and was therefore the antibody used in all subsequent experiments. α-AU13 was further characterized in native olfactory bulb membranes, where the appropriate molecular weight band was preabsorbed by incubation with the 46 amino acid peptide used to generate the antiserum (data not shown).

    Techniques: Immunoprecipitation, SDS Page, Western Blot, Expressing, Transformation Assay

    Callosal Kv1.3 channel protein in axons and glia is altered after injury and with CFZ treatment A. Confocal overlay showing Kv1.3 ( red ) and Kv1.2 ( green ) in rat corpus callosum 24h following midline fluid percussion TBI. Low magnification shows that each channel protein is found in reactive glia around callosal vessels (arrowheads) and along axon bundles (arrows). Inset shows paired paranodal distribution of Kv1.3 and Kv1.2 channels, some nodes with co-localization (yellow arrow), others with single channel expression (green, red arrows). B . Confocal overlays showing Kv1.3 in callosal astrocytes of sham injured (GFAP+, left-arrows; inset shows cell body and perivascular co-localization) and microglia of 24h postinjury cases (IBA1+, right-arrows). C. Western blot (WB) of protein extracts from 24h postinjury corpus callosum revealed that TBI reduced 67kD Kv1.3 levels and that CFZ treatment reversed loss of Kv1.3 expression. Data expressed as percent of paired untreated sham controls run on same blot. Lanes representative of group effects are shown in each panel. (ANOVA, *p

    Journal: Experimental neurology

    Article Title: Targeting Kv1.3 channels to reduce white matter pathology after traumatic brain injury

    doi: 10.1016/j.expneurol.2016.06.011

    Figure Lengend Snippet: Callosal Kv1.3 channel protein in axons and glia is altered after injury and with CFZ treatment A. Confocal overlay showing Kv1.3 ( red ) and Kv1.2 ( green ) in rat corpus callosum 24h following midline fluid percussion TBI. Low magnification shows that each channel protein is found in reactive glia around callosal vessels (arrowheads) and along axon bundles (arrows). Inset shows paired paranodal distribution of Kv1.3 and Kv1.2 channels, some nodes with co-localization (yellow arrow), others with single channel expression (green, red arrows). B . Confocal overlays showing Kv1.3 in callosal astrocytes of sham injured (GFAP+, left-arrows; inset shows cell body and perivascular co-localization) and microglia of 24h postinjury cases (IBA1+, right-arrows). C. Western blot (WB) of protein extracts from 24h postinjury corpus callosum revealed that TBI reduced 67kD Kv1.3 levels and that CFZ treatment reversed loss of Kv1.3 expression. Data expressed as percent of paired untreated sham controls run on same blot. Lanes representative of group effects are shown in each panel. (ANOVA, *p

    Article Snippet: Kv1.3 was shown to be highly expressed on inflammatory leukocyte infiltrates in brains from multiple sclerosis patients, and Kv1.3 was elevated in both gray and white matter ( ).

    Techniques: Expressing, Western Blot

    Corpus callosum mixed glial cultures grown in MatTek dishes and subjected to confocal dual labeling with antibodies to Kv1.3 (green) and microglial marker protein IBA1 (red) or astrocyte marker protein GFAP (red) A. Microglia are predominantly ramified in untreated cultures (arrowhead), shifting to reactive rounded cells with lobular processes after LPS (arrows). Kv1.3 protein is localized within the majority of microglia in each field and shifts from a normal concentration around nuclei (inset, UNT), to a more uniform distribution after inflammatory stimulation (inset, LPS). B. Astrocytes show mixed flat and fibrous morphologies. Kv1.3 signal is much reduced in astrocytes relative to surrounding microglia (not stained in these images) and predominantly found in small aggregates around cell nuclei (arrow). Astrocytes do not show LPS group differences in Kv1.3 expression. Bars= 20 µm.

    Journal: Experimental neurology

    Article Title: Targeting Kv1.3 channels to reduce white matter pathology after traumatic brain injury

    doi: 10.1016/j.expneurol.2016.06.011

    Figure Lengend Snippet: Corpus callosum mixed glial cultures grown in MatTek dishes and subjected to confocal dual labeling with antibodies to Kv1.3 (green) and microglial marker protein IBA1 (red) or astrocyte marker protein GFAP (red) A. Microglia are predominantly ramified in untreated cultures (arrowhead), shifting to reactive rounded cells with lobular processes after LPS (arrows). Kv1.3 protein is localized within the majority of microglia in each field and shifts from a normal concentration around nuclei (inset, UNT), to a more uniform distribution after inflammatory stimulation (inset, LPS). B. Astrocytes show mixed flat and fibrous morphologies. Kv1.3 signal is much reduced in astrocytes relative to surrounding microglia (not stained in these images) and predominantly found in small aggregates around cell nuclei (arrow). Astrocytes do not show LPS group differences in Kv1.3 expression. Bars= 20 µm.

    Article Snippet: Kv1.3 was shown to be highly expressed on inflammatory leukocyte infiltrates in brains from multiple sclerosis patients, and Kv1.3 was elevated in both gray and white matter ( ).

    Techniques: Labeling, Marker, Concentration Assay, Staining, Expressing