anti kv1 3  (Alomone Labs)


Bioz Verified Symbol Alomone Labs is a verified supplier
Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    Alomone Labs anti kv1 3
    Fyn modulates the posttranslational modification of <t>Kv1.3.</t> (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
    Anti Kv1 3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kv1 3/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv1 3 - by Bioz Stars, 2022-05
    95/100 stars

    Images

    1) 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

    2) 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

    3) 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:

    4) 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

    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 "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

    7) 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

    8) 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

    9) 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

    10) 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

    11) Product Images from "Inhibition of the potassium channel Kv1.3 reduces infarction and inflammation in ischemic stroke"

    Article Title: Inhibition of the potassium channel Kv1.3 reduces infarction and inflammation in ischemic stroke

    Journal: Annals of Clinical and Translational Neurology

    doi: 10.1002/acn3.513

    Kv1.3 inhibition does not affect phagocytosis in vivo. (A) Sections obtained 4 mm from the frontal pole on day‐8 after MCAO were stained for activated microglia/macrophages with CD 68 (green) and apoptosis associated TUNEL fragments (red). (B) Percentage of CD 68 + cells adjacent to or containing TUNEL + material. Three fields along the edge of the infarct area were analyzed in each section ( n = 3 animals per treatment group). Values are mean ± SD , P > 0.05. (C) Two‐dimensional image showing DAPI , CD 68 and TUNEL staining in a PAP ‐1 (10 mg/kg)‐treated mouse at higher magnification.
    Figure Legend Snippet: Kv1.3 inhibition does not affect phagocytosis in vivo. (A) Sections obtained 4 mm from the frontal pole on day‐8 after MCAO were stained for activated microglia/macrophages with CD 68 (green) and apoptosis associated TUNEL fragments (red). (B) Percentage of CD 68 + cells adjacent to or containing TUNEL + material. Three fields along the edge of the infarct area were analyzed in each section ( n = 3 animals per treatment group). Values are mean ± SD , P > 0.05. (C) Two‐dimensional image showing DAPI , CD 68 and TUNEL staining in a PAP ‐1 (10 mg/kg)‐treated mouse at higher magnification.

    Techniques Used: Inhibition, In Vivo, Staining, TUNEL Assay

    Kv1.3 expression in human infarcts. (A) Double immunofluorescent staining for activated microglia/macrophages ( MAC 387) and Kv1.3 in a roughly 2‐week‐old microinfarct. (B) Staining for Kv1.3 and iNOS in a needle biopsy from a 37‐year‐old man 7 days after an ischemic stroke.
    Figure Legend Snippet: Kv1.3 expression in human infarcts. (A) Double immunofluorescent staining for activated microglia/macrophages ( MAC 387) and Kv1.3 in a roughly 2‐week‐old microinfarct. (B) Staining for Kv1.3 and iNOS in a needle biopsy from a 37‐year‐old man 7 days after an ischemic stroke.

    Techniques Used: Expressing, Staining

    The Kv1.3 inhibitor PAP ‐1 reduces infarct area, improves neurological deficit, and reduces inflammatory cytokine production in mice. (A) NeuN‐defined infarct area in brain slices 2, 4, 6, and 8 mm from the frontal pole in vehicle‐treated mice or mice treated intraperitoneally with 10 mg/kg or 40 mg/kg PAP ‐1 twice daily started 12 h after reperfusion. (B) Neurological deficit in the 14‐score system (normal mouse = 14). (C) Brain cytokine concentrations in the ipsi‐ and contralateral side 8 days after MCAO from vehicle or PAP ‐1 (40 mg/kg)‐treated mice or shams ( n = 3). All values are mean ± S.E.M.
    Figure Legend Snippet: The Kv1.3 inhibitor PAP ‐1 reduces infarct area, improves neurological deficit, and reduces inflammatory cytokine production in mice. (A) NeuN‐defined infarct area in brain slices 2, 4, 6, and 8 mm from the frontal pole in vehicle‐treated mice or mice treated intraperitoneally with 10 mg/kg or 40 mg/kg PAP ‐1 twice daily started 12 h after reperfusion. (B) Neurological deficit in the 14‐score system (normal mouse = 14). (C) Brain cytokine concentrations in the ipsi‐ and contralateral side 8 days after MCAO from vehicle or PAP ‐1 (40 mg/kg)‐treated mice or shams ( n = 3). All values are mean ± S.E.M.

    Techniques Used: Mouse Assay

    Functional Kv1.3 channel expression on acutely isolated MCAO microglia. Representative whole‐cell current traces showing Kv1.3 currents recorded from microglia acutely isolated from either (A) normal control mouse brain, (B) day‐2 ipsilateral MCAO mouse brain or (C) day‐8 ipsilateral MCAO mouse brain. (D) Scatterplot showing Kv1.3 current amplitude measured from individual microglial cells for each condition. A significant increase in Kv1.3 current expression is observed starting on day‐2 (51.41 ± 29.24 pA , n = 12) and day‐5 (41.78 ± 26.49 pA , n = 19) on ipsilateral microglia compared to normal control (14.46 ± 8.60 pA , n = 15) and day‐8 contralateral microglia (15.16 ± 10.38 pA , n = 18). *** P
    Figure Legend Snippet: Functional Kv1.3 channel expression on acutely isolated MCAO microglia. Representative whole‐cell current traces showing Kv1.3 currents recorded from microglia acutely isolated from either (A) normal control mouse brain, (B) day‐2 ipsilateral MCAO mouse brain or (C) day‐8 ipsilateral MCAO mouse brain. (D) Scatterplot showing Kv1.3 current amplitude measured from individual microglial cells for each condition. A significant increase in Kv1.3 current expression is observed starting on day‐2 (51.41 ± 29.24 pA , n = 12) and day‐5 (41.78 ± 26.49 pA , n = 19) on ipsilateral microglia compared to normal control (14.46 ± 8.60 pA , n = 15) and day‐8 contralateral microglia (15.16 ± 10.38 pA , n = 18). *** P

    Techniques Used: Functional Assay, Expressing, Isolation

    Kv1.3 expression on microglia in the border zone of ischemic infarcts in mice. (A) Representative immunofluorescent staining showing Iba1 and Kv1.3 expression 2, 5 and 8 days after MCAO with reperfusion. (B) Kv1.3 and CD 68 or Kv1.3 and iNOS double staining 8 days after MCAO with reperfusion.
    Figure Legend Snippet: Kv1.3 expression on microglia in the border zone of ischemic infarcts in mice. (A) Representative immunofluorescent staining showing Iba1 and Kv1.3 expression 2, 5 and 8 days after MCAO with reperfusion. (B) Kv1.3 and CD 68 or Kv1.3 and iNOS double staining 8 days after MCAO with reperfusion.

    Techniques Used: Expressing, Mouse Assay, Staining, Double Staining

    Kv1.3 inhibition with PAP ‐1 reduces infarction and improves neurological deficit in rats. (A) Fluorescent staining for Kv1.3 and iNOS in the border zone of an ischemic infarct from a rat, which contains many ED 1 (= CD 68) positive microglia/macrophages. (B) Infarct area in brain slices 2, 4, 6, 8, 10, 12 and 16 mm from the frontal pole in vehicle‐treated rats or rats treated with 40 mg/kg PAP ‐1 twice daily started 12 h after reperfusion. (C) Neurological deficit in the 14‐score system (normal rat = 14). Values are mean ± S.E.M.
    Figure Legend Snippet: Kv1.3 inhibition with PAP ‐1 reduces infarction and improves neurological deficit in rats. (A) Fluorescent staining for Kv1.3 and iNOS in the border zone of an ischemic infarct from a rat, which contains many ED 1 (= CD 68) positive microglia/macrophages. (B) Infarct area in brain slices 2, 4, 6, 8, 10, 12 and 16 mm from the frontal pole in vehicle‐treated rats or rats treated with 40 mg/kg PAP ‐1 twice daily started 12 h after reperfusion. (C) Neurological deficit in the 14‐score system (normal rat = 14). Values are mean ± S.E.M.

    Techniques Used: Inhibition, Staining

    12) Product Images from "The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation"

    Article Title: The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.101497

    Effect of FS48 on the secretion of TNF-α and IL-2 in Jurkat T cells stimulated with PMA/ionomycin. A , effect of FS48 (1, 3, 10, 30 μM) and 100 nM MgTx on the proliferation of Jurkat T cells stimulated with PMA/ionomycin. B , knockdown of Kv1.3 channel expression with different siRNA. C and D , the mRNA production of TNF-α and IL-2. E and F , the inhibition rate of TNF-α and IL-2 secretion in Jurkat T cells. G and H , the inhibition rate of TNF-α and IL-2 in Jurkat T cells after knockdown Kv1.3. All data are presented as mean ± SD (n ≥ 3). ### p
    Figure Legend Snippet: Effect of FS48 on the secretion of TNF-α and IL-2 in Jurkat T cells stimulated with PMA/ionomycin. A , effect of FS48 (1, 3, 10, 30 μM) and 100 nM MgTx on the proliferation of Jurkat T cells stimulated with PMA/ionomycin. B , knockdown of Kv1.3 channel expression with different siRNA. C and D , the mRNA production of TNF-α and IL-2. E and F , the inhibition rate of TNF-α and IL-2 secretion in Jurkat T cells. G and H , the inhibition rate of TNF-α and IL-2 in Jurkat T cells after knockdown Kv1.3. All data are presented as mean ± SD (n ≥ 3). ### p

    Techniques Used: Expressing, Inhibition

    Effects of FS48 on mRNA and protein expression of Kv1.3 channel. A , the viability of Jurkat T cells incubated with indicated concentrations of FS48 for 24 h. B , the relative expression analysis of KCNA3 mRNA in the presence and absence of FS48 and MgTx by qRT-PCR. C , Kv1.3 protein expression analysis of Kv1.3 channel. The cells were treated with PMA/ionomycin (50 ng/ml; 1 μg/ml) for 24 h after incubated with 3 μM FS48 and 100 nM MgTx for 1 h and then were collected for Western blot analysis. D , the ratios of Kv1.3 proteins to GAPDH. Quantity One software (Bio-Rad) was used for band density analysis. Data are shown as mean ± SD (n ≥ 3). # p
    Figure Legend Snippet: Effects of FS48 on mRNA and protein expression of Kv1.3 channel. A , the viability of Jurkat T cells incubated with indicated concentrations of FS48 for 24 h. B , the relative expression analysis of KCNA3 mRNA in the presence and absence of FS48 and MgTx by qRT-PCR. C , Kv1.3 protein expression analysis of Kv1.3 channel. The cells were treated with PMA/ionomycin (50 ng/ml; 1 μg/ml) for 24 h after incubated with 3 μM FS48 and 100 nM MgTx for 1 h and then were collected for Western blot analysis. D , the ratios of Kv1.3 proteins to GAPDH. Quantity One software (Bio-Rad) was used for band density analysis. Data are shown as mean ± SD (n ≥ 3). # p

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Software

    Modulation of FS48 on endogenous voltage-gated potassium channels. A , representative traces of MgTx and different concentrations of FS48 suppressing the Kv1.3 currents in Jurkat T cells. Currents were elicited by applying 200 ms depolarization pulses from a holding potential of −70 mV to +40 mV in Jurkat T cells. B , concentration–response curve of FS48 inhibiting Kv1.3 currents in Jurkat T cells. Currents were normalized to the control and fitted by a Hill equation. C , current–voltage relationships (I-V). Test potentials were ranged from −50 mV to +40 mV with 10 mV increment steps. Y-axis represents the currents at different activation potential and normalized to the bath current at +40 mV in the present ( red ) or absent ( black ) of FS48; The solid lines represent the average Boltzmann sigmoidal fits. Data are shown as mean ± SD (n ≥ 3). ∗ p
    Figure Legend Snippet: Modulation of FS48 on endogenous voltage-gated potassium channels. A , representative traces of MgTx and different concentrations of FS48 suppressing the Kv1.3 currents in Jurkat T cells. Currents were elicited by applying 200 ms depolarization pulses from a holding potential of −70 mV to +40 mV in Jurkat T cells. B , concentration–response curve of FS48 inhibiting Kv1.3 currents in Jurkat T cells. Currents were normalized to the control and fitted by a Hill equation. C , current–voltage relationships (I-V). Test potentials were ranged from −50 mV to +40 mV with 10 mV increment steps. Y-axis represents the currents at different activation potential and normalized to the bath current at +40 mV in the present ( red ) or absent ( black ) of FS48; The solid lines represent the average Boltzmann sigmoidal fits. Data are shown as mean ± SD (n ≥ 3). ∗ p

    Techniques Used: Concentration Assay, Activation Assay

    13) Product Images from "Potassium secretion by voltage-gated potassium channel Kv1.3 in the rat kidney"

    Article Title: Potassium secretion by voltage-gated potassium channel Kv1.3 in the rat kidney

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00697.2009

    Kv1.3 expression in collecting duct intercalated cells from kidneys of HK-fed rats. Shown is detection of principal cells with Dolichus biflorus aggutinin coupled to fluorescein (DBA-F; A ; green), intercalated cells with goat-anti-B1-H-VATPase antibody ( D and G ; green), and Kv1.3 with an anti-Kv1.3 antibody ( B , E , and H ; red) in collecting ducts of HK-fed rats by indirect immunofluorescence confocal microscopy. Note that Kv1.3 is localized only to cells lacking DBA-F staining ( C ; merged) and in presumably intercalated cells expressing apical H + -VATPase ( F and I ; merged). Arrows point to apical Kv1.3-H + -VATPase colocalization (yellow). Bars = 25 μm.
    Figure Legend Snippet: Kv1.3 expression in collecting duct intercalated cells from kidneys of HK-fed rats. Shown is detection of principal cells with Dolichus biflorus aggutinin coupled to fluorescein (DBA-F; A ; green), intercalated cells with goat-anti-B1-H-VATPase antibody ( D and G ; green), and Kv1.3 with an anti-Kv1.3 antibody ( B , E , and H ; red) in collecting ducts of HK-fed rats by indirect immunofluorescence confocal microscopy. Note that Kv1.3 is localized only to cells lacking DBA-F staining ( C ; merged) and in presumably intercalated cells expressing apical H + -VATPase ( F and I ; merged). Arrows point to apical Kv1.3-H + -VATPase colocalization (yellow). Bars = 25 μm.

    Techniques Used: Expressing, Immunofluorescence, Confocal Microscopy, Staining

    Dietary K + intake has no effect on Kv1.3 channel transcript and protein expression. A : real-time PCR assays were performed to detect the relative abundance of Kv1.3 transcript in Cx, OM, and IM from rats fed control K + (CK) and high-K + (HK) diets. There was no significant difference in the Kv1.3 mRNA expression in any region between the CK and HK groups ( n = 6). B : representative Western blot showing Kv1.3 in crude membranes from Cx, OM, and IM from rats CK and HK diets. Crude protein (30 μg) was loaded on each lane. No differences were observed in Kv1.3 protein expression between the CK and HK groups.
    Figure Legend Snippet: Dietary K + intake has no effect on Kv1.3 channel transcript and protein expression. A : real-time PCR assays were performed to detect the relative abundance of Kv1.3 transcript in Cx, OM, and IM from rats fed control K + (CK) and high-K + (HK) diets. There was no significant difference in the Kv1.3 mRNA expression in any region between the CK and HK groups ( n = 6). B : representative Western blot showing Kv1.3 in crude membranes from Cx, OM, and IM from rats CK and HK diets. Crude protein (30 μg) was loaded on each lane. No differences were observed in Kv1.3 protein expression between the CK and HK groups.

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

    Effect of dietary K + intake on Kv1.3 expression in plasma membranes. A : representative immunoblot of Kv1.3 in plasma membranes from Cx, OM, and IM of rats fed CK and HK diets. Thirty micrograms of protein was loaded on each lane. B : significant relative increase was detected in the 3 regions of kidney studied in response to dietary K + loading, suggesting increased trafficking or stability of Kv1.3 protein to/on plasma membranes. Values are mean ± SE; n = 6/dietary group. * P
    Figure Legend Snippet: Effect of dietary K + intake on Kv1.3 expression in plasma membranes. A : representative immunoblot of Kv1.3 in plasma membranes from Cx, OM, and IM of rats fed CK and HK diets. Thirty micrograms of protein was loaded on each lane. B : significant relative increase was detected in the 3 regions of kidney studied in response to dietary K + loading, suggesting increased trafficking or stability of Kv1.3 protein to/on plasma membranes. Values are mean ± SE; n = 6/dietary group. * P

    Techniques Used: Expressing

    Immunohistochemistry of Kv1.3 in the kidney Cx in rats fed CK ( A ) and HK diets ( B ). Cortical collecting ducts (CCDs) are observed with positive cytoplasmic ( A ) and polarized ( B , arrows) immunoreactivity. Higher magnification shows a cytoplasmic distribution in all CCD cells of Kv1.3 in CK rats ( C ); the inset at the bottom left shows a negative control (preincubation of the anti-Kv1.3 antibody with the control peptide). The HK diet enhances apical expression of Kv1.3 in a few cells ( D , arrows). Bars = 50 μm.
    Figure Legend Snippet: Immunohistochemistry of Kv1.3 in the kidney Cx in rats fed CK ( A ) and HK diets ( B ). Cortical collecting ducts (CCDs) are observed with positive cytoplasmic ( A ) and polarized ( B , arrows) immunoreactivity. Higher magnification shows a cytoplasmic distribution in all CCD cells of Kv1.3 in CK rats ( C ); the inset at the bottom left shows a negative control (preincubation of the anti-Kv1.3 antibody with the control peptide). The HK diet enhances apical expression of Kv1.3 in a few cells ( D , arrows). Bars = 50 μm.

    Techniques Used: Immunohistochemistry, Negative Control, Expressing

    14) 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

    15) Product Images from "Expression of Kv1.3 potassium channels regulates density of cortical interneurons"

    Article Title: Expression of Kv1.3 potassium channels regulates density of cortical interneurons

    Journal: Developmental neurobiology

    doi: 10.1002/dneu.22105

    Different classes of cortical interneurons express Kv1.3 channels. Double immunofluorescence staining was used to determine the presence of Kv1.3 channels in the different types of interneurons tested. In every case interneurons are labeled with secondary
    Figure Legend Snippet: Different classes of cortical interneurons express Kv1.3 channels. Double immunofluorescence staining was used to determine the presence of Kv1.3 channels in the different types of interneurons tested. In every case interneurons are labeled with secondary

    Techniques Used: Double Immunofluorescence Staining, Labeling

    Deletion of Kv1.3 affects cortical interneuron numbers, densities and cortical volume. A. The mean number of cortical interneurons is always higher in controls than in Kv1.3 −/− except for PV in which the trend is reversed. Also, the difference
    Figure Legend Snippet: Deletion of Kv1.3 affects cortical interneuron numbers, densities and cortical volume. A. The mean number of cortical interneurons is always higher in controls than in Kv1.3 −/− except for PV in which the trend is reversed. Also, the difference

    Techniques Used:

    Deletion of Kv1.3 affects cortical interneuron numbers and densities
    Figure Legend Snippet: Deletion of Kv1.3 affects cortical interneuron numbers and densities

    Techniques Used:

    Expression of the transcription factor CDP (CCAAT displacement protein), a marker for layers II/III/IV, appears mostly absent in layers III/IV of Kv1.3−/− mice while expression of ER81 (layer 5 marker) and FoxP2 (layer 6 marker) appear
    Figure Legend Snippet: Expression of the transcription factor CDP (CCAAT displacement protein), a marker for layers II/III/IV, appears mostly absent in layers III/IV of Kv1.3−/− mice while expression of ER81 (layer 5 marker) and FoxP2 (layer 6 marker) appear

    Techniques Used: Expressing, Marker, Mouse Assay

    Deletion of Kv1.3 increases the number of PV interneurons in the cerebral cortex. The upper diagrams show actual samplings. The punctate marks inside of the cortex represent positively stained cells plotted inside counting boxes (200 μm ×
    Figure Legend Snippet: Deletion of Kv1.3 increases the number of PV interneurons in the cerebral cortex. The upper diagrams show actual samplings. The punctate marks inside of the cortex represent positively stained cells plotted inside counting boxes (200 μm ×

    Techniques Used: Staining

    Deletion of Kv1.3 decreases the number of SOM interneurons in the cerebral cortex. The upper diagrams show the actual samplings. The punctate marks inside of the cortex represent positively stained cells plotted inside counting boxes, which were the same
    Figure Legend Snippet: Deletion of Kv1.3 decreases the number of SOM interneurons in the cerebral cortex. The upper diagrams show the actual samplings. The punctate marks inside of the cortex represent positively stained cells plotted inside counting boxes, which were the same

    Techniques Used: Staining

    A. Kv1.3 stains the extracellular membrane of all cultured primary cortical cells (not permeabilized) that were visualized using the confocal microscope. Cell cultures were obtained at E14.5 and staining was done at E15.5. Kv1.3 is visualized with an
    Figure Legend Snippet: A. Kv1.3 stains the extracellular membrane of all cultured primary cortical cells (not permeabilized) that were visualized using the confocal microscope. Cell cultures were obtained at E14.5 and staining was done at E15.5. Kv1.3 is visualized with an

    Techniques Used: Cell Culture, Microscopy, Staining

    16) Product Images from "Overexpression of Delayed Rectifier K+ Channels Promotes In situ Proliferation of Leukocytes in Rat Kidneys with Advanced Chronic Renal Failure"

    Article Title: Overexpression of Delayed Rectifier K+ Channels Promotes In situ Proliferation of Leukocytes in Rat Kidneys with Advanced Chronic Renal Failure

    Journal: International Journal of Nephrology

    doi: 10.1155/2012/581581

    Kv1.3 and cell cycle marker expression in sham operated (sham) and advanced CRF rat kidneys. (A) Kv1.3 expression. (a) KCNA3 mRNA abundance in the renal cortex of sham operated (sham) and advanced CRF (CRF) rat kidneys. (b) and (c) Immunohistochemistry using antibody for Kv1.3 (brown) in sham operated and advanced CRF rat kidneys. High-power views of cortical interstitium. Magnification, ×60. (B) Cell cycle marker expression. The mRNA abundance of cyclin-dependent kinase 4 (Cdk4) (left) and p21 (right) in the renal cortex of sham operated and advanced CRF rat kidneys. # P
    Figure Legend Snippet: Kv1.3 and cell cycle marker expression in sham operated (sham) and advanced CRF rat kidneys. (A) Kv1.3 expression. (a) KCNA3 mRNA abundance in the renal cortex of sham operated (sham) and advanced CRF (CRF) rat kidneys. (b) and (c) Immunohistochemistry using antibody for Kv1.3 (brown) in sham operated and advanced CRF rat kidneys. High-power views of cortical interstitium. Magnification, ×60. (B) Cell cycle marker expression. The mRNA abundance of cyclin-dependent kinase 4 (Cdk4) (left) and p21 (right) in the renal cortex of sham operated and advanced CRF rat kidneys. # P

    Techniques Used: Marker, Expressing, Immunohistochemistry

    17) Product Images from "A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation"

    Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-017-0906-6

    Cartoon showing the proposed mechanism by which Kv1.3 channel function regulates LPS-induced EHD1 and TAP1 upregulation and MHC I trafficking an antigen presentation by microglia
    Figure Legend Snippet: Cartoon showing the proposed mechanism by which Kv1.3 channel function regulates LPS-induced EHD1 and TAP1 upregulation and MHC I trafficking an antigen presentation by microglia

    Techniques Used:

    Kv1.3 channels regulate TAP1, EHD1, GABPA, and IL1B expression in primary murine microglia. a Experimental plan for in vivo studies: adult C57BL/6 mice received four daily IP doses of PBS, ShK-223, LPS, or LPS+ShK-223, and brain mononuclear cells were isolated for flow cytometry from one hemisphere and qRT-PCR studies from the other hemisphere ( n = 3, mice/group). b Results from flow cytometric studies measuring intracellular EHD1 protein expression in freshly isolated CNS MPs (gated first on live cells, followed by CD11b + CD45 low and CD11b + CD45 high populations). At least 10,000 live CNS MPs were counted per sample. Left: Example of flow cytometric histograms comparing EHD1 expression in CD11b + CD45 low microglia from one mouse from each treatment group. Right: Quantitative analysis of EHD1 + cells in CD11b + CD45 low and CD11b + CD45 high populations. c Results from pRT-PCR studies measuring mRNA expression of TAP1, EHD1, GABPA, and IL1B in CNS MPs. For these studies, all CNS MPs isolated from one hemisphere were used for RNA extraction in Trizol, followed by cDNA preparation followed by quantitative PCR. HPRT was used as the housekeeping gene. Data normalized to HPRT were then normalized to PBS-treated control samples (* p
    Figure Legend Snippet: Kv1.3 channels regulate TAP1, EHD1, GABPA, and IL1B expression in primary murine microglia. a Experimental plan for in vivo studies: adult C57BL/6 mice received four daily IP doses of PBS, ShK-223, LPS, or LPS+ShK-223, and brain mononuclear cells were isolated for flow cytometry from one hemisphere and qRT-PCR studies from the other hemisphere ( n = 3, mice/group). b Results from flow cytometric studies measuring intracellular EHD1 protein expression in freshly isolated CNS MPs (gated first on live cells, followed by CD11b + CD45 low and CD11b + CD45 high populations). At least 10,000 live CNS MPs were counted per sample. Left: Example of flow cytometric histograms comparing EHD1 expression in CD11b + CD45 low microglia from one mouse from each treatment group. Right: Quantitative analysis of EHD1 + cells in CD11b + CD45 low and CD11b + CD45 high populations. c Results from pRT-PCR studies measuring mRNA expression of TAP1, EHD1, GABPA, and IL1B in CNS MPs. For these studies, all CNS MPs isolated from one hemisphere were used for RNA extraction in Trizol, followed by cDNA preparation followed by quantitative PCR. HPRT was used as the housekeeping gene. Data normalized to HPRT were then normalized to PBS-treated control samples (* p

    Techniques Used: Expressing, In Vivo, Mouse Assay, Isolation, Flow Cytometry, Quantitative RT-PCR, Polymerase Chain Reaction, RNA Extraction, Real-time Polymerase Chain Reaction

    Identification of Kv1.3-regulated signaling mechanisms in LPS-induced microglial activation. a , b Canonical pathway analysis of 120 Kv1.3-dependent proteins identified in our proteomic dataset revealed highly represented signaling pathways, two of which are shown here (see Additional file 2 : Table S5 for others). Members of this list of 120 proteins are marked with ( red circle ). Transcription factors are also highlighted ( transparent red circle ). Arrows indicate directionality of the interaction (upstream vs. downstream). a GABPA, a Kv1.3-dependent transcription factor that was downregulated by LPS, was placed upstream of several Kv1.3-regulated proteins. b MHCI proteins of relevance to our results (TAP1, Tapasin, and EHD1 proteins) were represented in a signaling network that suggested that STAT1 and IRF1 may serve as upstream regulators of these proteins. c Comparison of normalized protein expression of transcription factors identified in our proteomic dataset across treatment groups. d Quantitative RT-PCR data comparing IRF1, IRF7, and NFKB1 mRNA expression across treatment groups (paired t tests were used for these comparisons; six replicates/group). e Phospho-flow cytometric studies of serine (S727) and tyrosine (Y701) STAT1 phosphorylation in BV2 microglia ( n = 5/treatment group) at 30-min and 3-h time points. f Immunofluorescence microscopy showing partial co-localization between Kv1.3 ( green , detected by ShK-F6CA labeling) and CD14 ( red ) in LPS-activated BV2 microglia (* p
    Figure Legend Snippet: Identification of Kv1.3-regulated signaling mechanisms in LPS-induced microglial activation. a , b Canonical pathway analysis of 120 Kv1.3-dependent proteins identified in our proteomic dataset revealed highly represented signaling pathways, two of which are shown here (see Additional file 2 : Table S5 for others). Members of this list of 120 proteins are marked with ( red circle ). Transcription factors are also highlighted ( transparent red circle ). Arrows indicate directionality of the interaction (upstream vs. downstream). a GABPA, a Kv1.3-dependent transcription factor that was downregulated by LPS, was placed upstream of several Kv1.3-regulated proteins. b MHCI proteins of relevance to our results (TAP1, Tapasin, and EHD1 proteins) were represented in a signaling network that suggested that STAT1 and IRF1 may serve as upstream regulators of these proteins. c Comparison of normalized protein expression of transcription factors identified in our proteomic dataset across treatment groups. d Quantitative RT-PCR data comparing IRF1, IRF7, and NFKB1 mRNA expression across treatment groups (paired t tests were used for these comparisons; six replicates/group). e Phospho-flow cytometric studies of serine (S727) and tyrosine (Y701) STAT1 phosphorylation in BV2 microglia ( n = 5/treatment group) at 30-min and 3-h time points. f Immunofluorescence microscopy showing partial co-localization between Kv1.3 ( green , detected by ShK-F6CA labeling) and CD14 ( red ) in LPS-activated BV2 microglia (* p

    Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Microscopy, Labeling

    LPS-induced MHCI trafficking in microglia involves Kv1.3 channels. a Comparison of MHCI and MHCI-related protein expression in BV2 proteomic data. Normalized LFQ data (compared to control-treatment) is shown (three replicates/group). b Experimental plan for in vivo studies: Adult C57BL/6 mice received four daily IP doses of PBS, ShK-223, LPS, or LPS+ShK-223 and brain mononuclear cells were isolated for flow cytometry (pooled n = 9/group). c Comparison of MHCI (H2Kb) expression (median fluorescence intensity) in CD11b + CD45 low microglia. d Representative flow cytometry frequency histograms of MHCI expression in CD11b + CD45 low microglia. e Comparison of MHCI expression in CD11b + CD45 high brain-infiltrating macrophages. f Comparison of the proportions of CD11b + CD45 high CNS macrophages (among all CD11b + cells) in the four treatment groups (* p
    Figure Legend Snippet: LPS-induced MHCI trafficking in microglia involves Kv1.3 channels. a Comparison of MHCI and MHCI-related protein expression in BV2 proteomic data. Normalized LFQ data (compared to control-treatment) is shown (three replicates/group). b Experimental plan for in vivo studies: Adult C57BL/6 mice received four daily IP doses of PBS, ShK-223, LPS, or LPS+ShK-223 and brain mononuclear cells were isolated for flow cytometry (pooled n = 9/group). c Comparison of MHCI (H2Kb) expression (median fluorescence intensity) in CD11b + CD45 low microglia. d Representative flow cytometry frequency histograms of MHCI expression in CD11b + CD45 low microglia. e Comparison of MHCI expression in CD11b + CD45 high brain-infiltrating macrophages. f Comparison of the proportions of CD11b + CD45 high CNS macrophages (among all CD11b + cells) in the four treatment groups (* p

    Techniques Used: Expressing, In Vivo, Mouse Assay, Isolation, Flow Cytometry, Fluorescence

    Kv1.3 blockade inhibits LPS-induced MHCI-restricted antigen presentation to CD8 T cells. a Experimental plan for MHCI-restricted T cell proliferation studies: BV2 microglia were treated in vitro with PBS, ShK-223 (100 nM), LPS (100 ng/mL), or LPS+ShK-223 for 24 h (four replicates/group) and then loaded with 2 μg/mL Ova (257-264) peptide for 30 min at 37 °C. Washed microglia were co-cultured with CFSE-labeled splenic T cells from Ova (257-264)-specific OT-1 mice for 48 h (25,000 microglia:150,000 T cells/well). For in vivo experiments, adult wild-type mice were treated with four daily IP doses of saline, ShK-223, LPS, or LPS+ShK-223 ( n = 6/group) and isolated brain mononuclear cells were used for in vitro T cell proliferation studies. After 48 h, CFSE dilution (leftward shift in CFSE staining) was assessed by flow cytometry as a measure of CD8 + T cell proliferation. b Comparison of proliferating CD8 + cells across various treatment groups from in vitro and in vivo studies. Right : Representative frequency histograms from in vivo experiments. No direct effects of LPS or Ova peptide on T cells were noted without the presence of microglia. c Flow cytometric detection of CD3 + CD11 b − T cells in brain mononuclear cells after thorough cardiac perfusion. Mice were injected (IP, once daily for 4 days) with PBS, LPS, ShK-223, or LPS+ShK-223. Panel a shows the gating strategy. After forward and side-scatter gating for live mononuclear cell population, CD11b − CD3 + cells were gated and assessed for T cell markers (CD3, CD8) and activation/cytolytic marker CD95 (Fas). d Flow cytometric evidence for increased CD8 + T cell trafficking to the brain following LPS treatment. e Comparison of proportions of brain CD8 + and CD8 − T cell populations across the 4 treatment groups. f Comparison of CD95 (Fas) expression in brain CD8 + and CD8 − T cell populations across treatment groups (* p
    Figure Legend Snippet: Kv1.3 blockade inhibits LPS-induced MHCI-restricted antigen presentation to CD8 T cells. a Experimental plan for MHCI-restricted T cell proliferation studies: BV2 microglia were treated in vitro with PBS, ShK-223 (100 nM), LPS (100 ng/mL), or LPS+ShK-223 for 24 h (four replicates/group) and then loaded with 2 μg/mL Ova (257-264) peptide for 30 min at 37 °C. Washed microglia were co-cultured with CFSE-labeled splenic T cells from Ova (257-264)-specific OT-1 mice for 48 h (25,000 microglia:150,000 T cells/well). For in vivo experiments, adult wild-type mice were treated with four daily IP doses of saline, ShK-223, LPS, or LPS+ShK-223 ( n = 6/group) and isolated brain mononuclear cells were used for in vitro T cell proliferation studies. After 48 h, CFSE dilution (leftward shift in CFSE staining) was assessed by flow cytometry as a measure of CD8 + T cell proliferation. b Comparison of proliferating CD8 + cells across various treatment groups from in vitro and in vivo studies. Right : Representative frequency histograms from in vivo experiments. No direct effects of LPS or Ova peptide on T cells were noted without the presence of microglia. c Flow cytometric detection of CD3 + CD11 b − T cells in brain mononuclear cells after thorough cardiac perfusion. Mice were injected (IP, once daily for 4 days) with PBS, LPS, ShK-223, or LPS+ShK-223. Panel a shows the gating strategy. After forward and side-scatter gating for live mononuclear cell population, CD11b − CD3 + cells were gated and assessed for T cell markers (CD3, CD8) and activation/cytolytic marker CD95 (Fas). d Flow cytometric evidence for increased CD8 + T cell trafficking to the brain following LPS treatment. e Comparison of proportions of brain CD8 + and CD8 − T cell populations across the 4 treatment groups. f Comparison of CD95 (Fas) expression in brain CD8 + and CD8 − T cell populations across treatment groups (* p

    Techniques Used: In Vitro, Cell Culture, Labeling, Mouse Assay, In Vivo, Isolation, Staining, Flow Cytometry, Injection, Activation Assay, Marker, Expressing

    LPS-activated BV2 microglia upregulate Kv1.3 channels. a Immunofluorescence microscopic comparison of Kv1.3 protein expression (detected by Anti-Kv1.3 rabbit pAb 1:500) by unstimulated and LPS-activated (100 ng/mL × 24 h) BV2 microglia. b Western blot confirmation of LPS dose-dependent increase in Kv1.3 protein in BV2 whole-cell lysates (Anti-Kv1.3 pAb 1:1000). c Immunofluorescence microscopic comparison of cell surface Kv1.3 channels in unstimulated and LPS-activated BV2 cells using ShK-F6CA. d Flow cytometric comparison of ShK-F6CA-labeled cell surface Kv1.3 channels and microglial activation marker ICAM-1 in unstimulated and LPS-activated BV2 cells. e Assessment of microglial activation markers MHCII and CD69 in unstimulated and LPS-activated BV2 cells
    Figure Legend Snippet: LPS-activated BV2 microglia upregulate Kv1.3 channels. a Immunofluorescence microscopic comparison of Kv1.3 protein expression (detected by Anti-Kv1.3 rabbit pAb 1:500) by unstimulated and LPS-activated (100 ng/mL × 24 h) BV2 microglia. b Western blot confirmation of LPS dose-dependent increase in Kv1.3 protein in BV2 whole-cell lysates (Anti-Kv1.3 pAb 1:1000). c Immunofluorescence microscopic comparison of cell surface Kv1.3 channels in unstimulated and LPS-activated BV2 cells using ShK-F6CA. d Flow cytometric comparison of ShK-F6CA-labeled cell surface Kv1.3 channels and microglial activation marker ICAM-1 in unstimulated and LPS-activated BV2 cells. e Assessment of microglial activation markers MHCII and CD69 in unstimulated and LPS-activated BV2 cells

    Techniques Used: Immunofluorescence, Expressing, Western Blot, Labeling, Activation Assay, Marker

    In vivo demonstration of Kv1.3 channel upregulation by microglia and brain-infiltrating macrophages in a mouse model of LPS-induced neuroinflammation. a LPS was administered to adult C57BL/6 mice by tail vein injection, and brain mononuclear cells were analyzed by flow cytometry (CD11b, CD45, ShK-F6CA, and ICAM-1). b Most cells were CD11b + CD45 low microglia while small populations of brain-infiltrating macrophages CD11b + CD45 high and CD11b - non-myeloid cells were observed. c – e Comparison of Kv1.3 channel and ICAM-1 expression in brain mononuclear cells isolated from control and LPS-treated mice in microglia ( c ), brain-infiltrating macrophages ( d ), and CD11b - subpopulations ( e ) (* p
    Figure Legend Snippet: In vivo demonstration of Kv1.3 channel upregulation by microglia and brain-infiltrating macrophages in a mouse model of LPS-induced neuroinflammation. a LPS was administered to adult C57BL/6 mice by tail vein injection, and brain mononuclear cells were analyzed by flow cytometry (CD11b, CD45, ShK-F6CA, and ICAM-1). b Most cells were CD11b + CD45 low microglia while small populations of brain-infiltrating macrophages CD11b + CD45 high and CD11b - non-myeloid cells were observed. c – e Comparison of Kv1.3 channel and ICAM-1 expression in brain mononuclear cells isolated from control and LPS-treated mice in microglia ( c ), brain-infiltrating macrophages ( d ), and CD11b - subpopulations ( e ) (* p

    Techniques Used: In Vivo, Mouse Assay, Injection, Flow Cytometry, Expressing, Isolation

    Kv1.3 channels regulate microglial taxis and formation of F-actin complexes induced by LPS. a In the gap closure assay of microglial taxis, BV2 cells were grown to near confluence followed by placement of a uniform scratch using a 200-μm pipette tip. Ability of microglia to close this gap was assessed by measuring the percentage of gap closure over a 24-h period. Paired (0 and 24 h) representative images from each treatment group are shown ( a , left ), and the comparison of percentage gap closure (over 24 h) is shown ( a , right ) (six replicates per condition). b BV2 microglial transmigration across a transwell membrane (8-μm pore diameter) after exposure to control, ShK-223, ShK-186, LPS or LPS+ShK-223, or ShK-186, towards serum-containing medium (10% fetal bovine serum). Following 24 h of transmigration, cells were detached from undersurface of the insert (0.25% Trypsin), and cells that successfully migrated across the membrane were counted on a hemocytometer ( n = 3, independent experiments). c Comparison of F-actin containing focal adhesion complexes in BV2 microglia following exposure to control, ShK-223, LPS, or LPS+ShK-223. Fixed and permeabilized BV2 cells were labeled with phalloidin-rhodamine to detect F-actin ( left : immunofluorescence images). The number of focal complexes were counted per cell at ×40 magnification ( right ) and compared ( > 25 cells counted per condition). d DCFDA assay of ROS production by brain mononuclear cells isolated from C57BL/6 mice treated with PBS, LPS, ShK-223, or LPS+ShK-223 IP for four consecutive days ( n = 3, mice/group). Cells were loaded with DCFDA for 30 min and assayed for ROS activity by flow cytometry. e Flow cytometric phagocytosis assay of fluorescent (PE) microbeads by brain mononuclear cells isolated from C57B6/L mice treated with PBS, LPS, ShK-223, or LPS+ShK-223, n = 3/group). Dotted line : fluorescence of cells not exposed to beads; gray histogram : PBS-treated; black histogram : LPS-treated. The proportions of all phagocytic cells and highly phagocytic cells were compared across treatment groups (* p
    Figure Legend Snippet: Kv1.3 channels regulate microglial taxis and formation of F-actin complexes induced by LPS. a In the gap closure assay of microglial taxis, BV2 cells were grown to near confluence followed by placement of a uniform scratch using a 200-μm pipette tip. Ability of microglia to close this gap was assessed by measuring the percentage of gap closure over a 24-h period. Paired (0 and 24 h) representative images from each treatment group are shown ( a , left ), and the comparison of percentage gap closure (over 24 h) is shown ( a , right ) (six replicates per condition). b BV2 microglial transmigration across a transwell membrane (8-μm pore diameter) after exposure to control, ShK-223, ShK-186, LPS or LPS+ShK-223, or ShK-186, towards serum-containing medium (10% fetal bovine serum). Following 24 h of transmigration, cells were detached from undersurface of the insert (0.25% Trypsin), and cells that successfully migrated across the membrane were counted on a hemocytometer ( n = 3, independent experiments). c Comparison of F-actin containing focal adhesion complexes in BV2 microglia following exposure to control, ShK-223, LPS, or LPS+ShK-223. Fixed and permeabilized BV2 cells were labeled with phalloidin-rhodamine to detect F-actin ( left : immunofluorescence images). The number of focal complexes were counted per cell at ×40 magnification ( right ) and compared ( > 25 cells counted per condition). d DCFDA assay of ROS production by brain mononuclear cells isolated from C57BL/6 mice treated with PBS, LPS, ShK-223, or LPS+ShK-223 IP for four consecutive days ( n = 3, mice/group). Cells were loaded with DCFDA for 30 min and assayed for ROS activity by flow cytometry. e Flow cytometric phagocytosis assay of fluorescent (PE) microbeads by brain mononuclear cells isolated from C57B6/L mice treated with PBS, LPS, ShK-223, or LPS+ShK-223, n = 3/group). Dotted line : fluorescence of cells not exposed to beads; gray histogram : PBS-treated; black histogram : LPS-treated. The proportions of all phagocytic cells and highly phagocytic cells were compared across treatment groups (* p

    Techniques Used: Transferring, Transmigration Assay, Labeling, Immunofluorescence, Isolation, Mouse Assay, Activity Assay, Flow Cytometry, Phagocytosis Assay, Fluorescence

    Identification of novel Kv1.3 channel-dependent molecular mechanisms by microglial quantitative proteomics. BV2 microglia were exposed to PBS, LPS (100 ng/mL), ShK-223 (100 nM), or LPS+ShK-223 for 24 h (3 replicates/group) and whole-cell lysates were used for mass-spectrometric analysis. a Volcano-plot: Of 450 proteins differentially expressed across all 4 groups ( black dots ), 144 proteins were significantly up- or downregulated following LPS exposure ( top-right and top-left quadrants; vertical dotted lines represent the upper and lower boundaries of 1.25-fold change threshold while the horizontal dotted line represents p value threshold of 0.05 comparing LPS vs. control groups). Among these, ShK-223 reversed the LPS effect in 21 proteins (highlighted in red ). b LPS-upregulated proteins that were reversed by ShK-223 and c LPS-downregulated proteins reversed by ShK-223 are shown. Relative expression represents label-free quantitation data in each treatment condition normalized to the control PBS group. The dotted horizontal line represents the twofold change threshold as compared to the PBS group. d – f Circle plots representing results from GO analyses are shown ( gray : protein/gene symbol, red : biological process, green : cellular component, purple : molecular function). Only significantly enriched GO terms (unadjusted p ≤ 0.05 based on enrichment score) are shown (* p
    Figure Legend Snippet: Identification of novel Kv1.3 channel-dependent molecular mechanisms by microglial quantitative proteomics. BV2 microglia were exposed to PBS, LPS (100 ng/mL), ShK-223 (100 nM), or LPS+ShK-223 for 24 h (3 replicates/group) and whole-cell lysates were used for mass-spectrometric analysis. a Volcano-plot: Of 450 proteins differentially expressed across all 4 groups ( black dots ), 144 proteins were significantly up- or downregulated following LPS exposure ( top-right and top-left quadrants; vertical dotted lines represent the upper and lower boundaries of 1.25-fold change threshold while the horizontal dotted line represents p value threshold of 0.05 comparing LPS vs. control groups). Among these, ShK-223 reversed the LPS effect in 21 proteins (highlighted in red ). b LPS-upregulated proteins that were reversed by ShK-223 and c LPS-downregulated proteins reversed by ShK-223 are shown. Relative expression represents label-free quantitation data in each treatment condition normalized to the control PBS group. The dotted horizontal line represents the twofold change threshold as compared to the PBS group. d – f Circle plots representing results from GO analyses are shown ( gray : protein/gene symbol, red : biological process, green : cellular component, purple : molecular function). Only significantly enriched GO terms (unadjusted p ≤ 0.05 based on enrichment score) are shown (* p

    Techniques Used: Expressing, Quantitation Assay

    18) 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

    19) Product Images from "Potassium Channel Kv1.3 Is Highly Expressed by Microglia in Human Alzheimer’s Disease"

    Article Title: Potassium Channel Kv1.3 Is Highly Expressed by Microglia in Human Alzheimer’s Disease

    Journal: Journal of Alzheimer's disease : JAD

    doi: 10.3233/JAD-141704

    Kv1.3 positive cells are found around Aβ aggregates in AD brain. A, B) Kv1.3 positive cells (brown) aggregate around Aβ plaques (blue-black). Arrows indicate Kv1.3 positive cells (A: 10×, B: 20×). C, D) Iba1 positive microglia,
    Figure Legend Snippet: Kv1.3 positive cells are found around Aβ aggregates in AD brain. A, B) Kv1.3 positive cells (brown) aggregate around Aβ plaques (blue-black). Arrows indicate Kv1.3 positive cells (A: 10×, B: 20×). C, D) Iba1 positive microglia,

    Techniques Used:

    Iba1 and Kv1.3 expression is increased in AD brains as compared to control brains. Left: Representative 20× images demonstrating low Iba1 and minimal Kv1.3 staining in a control brain (top panel) compared to strong Iba1 positivity and robust Kv1.3
    Figure Legend Snippet: Iba1 and Kv1.3 expression is increased in AD brains as compared to control brains. Left: Representative 20× images demonstrating low Iba1 and minimal Kv1.3 staining in a control brain (top panel) compared to strong Iba1 positivity and robust Kv1.3

    Techniques Used: Expressing, Staining

    Kv1.3 expression in frontal cortex of AD and non-AD controls as measured by immunoblotting. Kv1.3 expression was higher in the membrane fraction of AD brains ( n = 4) as compared to non-AD controls ( n = 5).
    Figure Legend Snippet: Kv1.3 expression in frontal cortex of AD and non-AD controls as measured by immunoblotting. Kv1.3 expression was higher in the membrane fraction of AD brains ( n = 4) as compared to non-AD controls ( n = 5).

    Techniques Used: Expressing

    Patterns of Kv1.3 channel expression in AD brains. Two staining patterns were observed. A) Kv1.3 staining in AD brain: “glial pattern” (20×). B) Secondary antibody control (20×). C) Kv1.3 staining in AD brain: “plaque-like”
    Figure Legend Snippet: Patterns of Kv1.3 channel expression in AD brains. Two staining patterns were observed. A) Kv1.3 staining in AD brain: “glial pattern” (20×). B) Secondary antibody control (20×). C) Kv1.3 staining in AD brain: “plaque-like”

    Techniques Used: Expressing, Staining

    Specificity of Kv1.3 antibody. Pre-adsorption with Kv1.3 immunizing peptide (8µg/ml) abolishes glial (A) as well as plaque-patterns (B) of Kv1.3 staining in frontal cortical regions of AD brain tissue.
    Figure Legend Snippet: Specificity of Kv1.3 antibody. Pre-adsorption with Kv1.3 immunizing peptide (8µg/ml) abolishes glial (A) as well as plaque-patterns (B) of Kv1.3 staining in frontal cortical regions of AD brain tissue.

    Techniques Used: Adsorption, Staining

    Kv1.3 channels in AD brains are predominantly expressed by Iba1 positive microglia. A) Kv1.3 (red) co-localizes robustly with Iba1 (green) [top panel] . B) Low levels of co-localization were observed between Kv1.3 (red) and GFAP (green) [bottom panel] .
    Figure Legend Snippet: Kv1.3 channels in AD brains are predominantly expressed by Iba1 positive microglia. A) Kv1.3 (red) co-localizes robustly with Iba1 (green) [top panel] . B) Low levels of co-localization were observed between Kv1.3 (red) and GFAP (green) [bottom panel] .

    Techniques Used:

    20) Product Images from "The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain"

    Article Title: The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0501770102

    Expression of Kv1.3 on inflammatory cells in MS brain. Paraffin sections were stained by indirect immunoperoxidase for Kv1.3, CD3, CD4, CCR7, and CCR5. Areas used in sectioning were from a white matter plaque. There were many perivascular inflammatory cells that stained positively for CD3 ( A ), Kv1.3 ( B ), and CD4 ( B Inset ) on consecutive sections. ( C ) Kv1.3 was also localized on inflammatory cells in the white matter parenchyma ( Inset reveals membrane polarization of Kv1.3 staining). ( D ) Consecutive sections through another perivascular infiltrate revealed numerous Kv1.3 + inflammatory cells, which were predominantly CCR7 - ( E ) ( Inset reveals rare CCR7 positive staining), and CCR5 + ( F ). (Scale bar, 50 μmin A and B and 20 μmin C , D , E , and F .)
    Figure Legend Snippet: Expression of Kv1.3 on inflammatory cells in MS brain. Paraffin sections were stained by indirect immunoperoxidase for Kv1.3, CD3, CD4, CCR7, and CCR5. Areas used in sectioning were from a white matter plaque. There were many perivascular inflammatory cells that stained positively for CD3 ( A ), Kv1.3 ( B ), and CD4 ( B Inset ) on consecutive sections. ( C ) Kv1.3 was also localized on inflammatory cells in the white matter parenchyma ( Inset reveals membrane polarization of Kv1.3 staining). ( D ) Consecutive sections through another perivascular infiltrate revealed numerous Kv1.3 + inflammatory cells, which were predominantly CCR7 - ( E ) ( Inset reveals rare CCR7 positive staining), and CCR5 + ( F ). (Scale bar, 50 μmin A and B and 20 μmin C , D , E , and F .)

    Techniques Used: Expressing, Mass Spectrometry, Staining

    Immunofluorescence and electrophysiology of peripheral and CSF T cells. FACS plots revealed staining patterns of CCR7 vs. CD45RA. ( A ) CD4-gated MS-derived CSF cells revealed a predominantly T CM phenotype immediately ex vivo , but with rapid conversion to short-lived T EM after 7 and 14 days of stimulation using anti-CD3/CD28 coated beads ( B and C ). By comparison, negatively sorted CSF derived from noninflammatory neurological controls showed CD4 + cells that were predominantly T CM ( D ) and remained T CM at day 7 despite identical manner of stimulation ( E ). ( F-H ) CD4 cells derived from PB were predominantly T CM and naive phenotype ( F ), and maintained high levels of CCR7 even after 7 and 14 days of stimulation using anti-CD3/CD28 coated beads ( G and H ). ( I ) Single cell patch-clamp analysis of MS-derived CSF cells revealed high Kv1.3 channel numbers per cell; mean channel number per cell was 1,082 ± 489 (mean ± SEM, n = 28). Shown for comparison on the right are mean Kv1.3 channel numbers from activated PB T cells from three MS patients ( n = 32), and from 10 healthy controls ( n = 33). ( J and K ) Family of currents. The test potential was changed from -60 to 60 mV in 10-mV increments every 30 s ( V 1/2 = -28 mV). ( L ) The current in CSF cells exhibited the characteristic use-dependence of Kv1.3 when 200-ms pulses were applied every second to 40 mV. ( M and N ) confirmed that the current is carried by Kv1.3. ( O ) Representative images of CSF resting (top) or activated (bottom) T cells stained with anti-Kv1.3 and CCR7 Abs. ( P ) Staining intensities for Kv1.3 and CCR7 in resting and activated CSF T cells. ( Q ) Z-stack of confocal images through an activated CSF T cell.
    Figure Legend Snippet: Immunofluorescence and electrophysiology of peripheral and CSF T cells. FACS plots revealed staining patterns of CCR7 vs. CD45RA. ( A ) CD4-gated MS-derived CSF cells revealed a predominantly T CM phenotype immediately ex vivo , but with rapid conversion to short-lived T EM after 7 and 14 days of stimulation using anti-CD3/CD28 coated beads ( B and C ). By comparison, negatively sorted CSF derived from noninflammatory neurological controls showed CD4 + cells that were predominantly T CM ( D ) and remained T CM at day 7 despite identical manner of stimulation ( E ). ( F-H ) CD4 cells derived from PB were predominantly T CM and naive phenotype ( F ), and maintained high levels of CCR7 even after 7 and 14 days of stimulation using anti-CD3/CD28 coated beads ( G and H ). ( I ) Single cell patch-clamp analysis of MS-derived CSF cells revealed high Kv1.3 channel numbers per cell; mean channel number per cell was 1,082 ± 489 (mean ± SEM, n = 28). Shown for comparison on the right are mean Kv1.3 channel numbers from activated PB T cells from three MS patients ( n = 32), and from 10 healthy controls ( n = 33). ( J and K ) Family of currents. The test potential was changed from -60 to 60 mV in 10-mV increments every 30 s ( V 1/2 = -28 mV). ( L ) The current in CSF cells exhibited the characteristic use-dependence of Kv1.3 when 200-ms pulses were applied every second to 40 mV. ( M and N ) confirmed that the current is carried by Kv1.3. ( O ) Representative images of CSF resting (top) or activated (bottom) T cells stained with anti-Kv1.3 and CCR7 Abs. ( P ) Staining intensities for Kv1.3 and CCR7 in resting and activated CSF T cells. ( Q ) Z-stack of confocal images through an activated CSF T cell.

    Techniques Used: Immunofluorescence, FACS, Staining, Mass Spectrometry, Derivative Assay, Ex Vivo, Patch Clamp

    Confocal microscopy of Kv1.3 and CD3/CD4 in MS brain tissue. Confocal microscopic images of 20-μm floating sections of postmortem MS brain tissue. ( A and B ) Parenchymal infiltrate with positive CD3 and punctate Kv1.3 staining in the membrane. ( C ) A cluster of lymphocytes stained positively for Kv1.3 and CD4, as well as occasional CD8 ( Inset ). ( D ) CD3 + cells showed extensive Kv1.3 and CD3 colocalization in the membrane. Immunofluorescent staining of PB-derived T cells concentrated on a slide by cytospin showed membrane patterns of staining of CD4, CD8, and Kv1.3. ( E ) Day 7-activated naive and T CM are CD4 + , CCR7 + (data not shown) and Kv1.3 - .( F ) Chronically stimulated resting T EM were CD4 + , CCR7 - (data not shown) and expressed Kv1.3 in the membrane but with minimal colocalization with CD4. ( G )T EM that had been recently activated expressed increased amounts of Kv1.3 and demonstrated more colocalization with CD4 similar to the brain tissue lymphocyte in D . ( H ) Naive CD8 + T cells expressed no Kv1.3 but, when chronically stimulated and activated ( I ), exhibited intense Kv1.3 expression and colocalization with CD8. ( J ) Isotype controls using nonspecific rabbit primary antibody followed by usual secondary label and with primary rabbit anti-human and nonspecific labeled mouse anti-rabbit secondary antibody showed no background staining.
    Figure Legend Snippet: Confocal microscopy of Kv1.3 and CD3/CD4 in MS brain tissue. Confocal microscopic images of 20-μm floating sections of postmortem MS brain tissue. ( A and B ) Parenchymal infiltrate with positive CD3 and punctate Kv1.3 staining in the membrane. ( C ) A cluster of lymphocytes stained positively for Kv1.3 and CD4, as well as occasional CD8 ( Inset ). ( D ) CD3 + cells showed extensive Kv1.3 and CD3 colocalization in the membrane. Immunofluorescent staining of PB-derived T cells concentrated on a slide by cytospin showed membrane patterns of staining of CD4, CD8, and Kv1.3. ( E ) Day 7-activated naive and T CM are CD4 + , CCR7 + (data not shown) and Kv1.3 - .( F ) Chronically stimulated resting T EM were CD4 + , CCR7 - (data not shown) and expressed Kv1.3 in the membrane but with minimal colocalization with CD4. ( G )T EM that had been recently activated expressed increased amounts of Kv1.3 and demonstrated more colocalization with CD4 similar to the brain tissue lymphocyte in D . ( H ) Naive CD8 + T cells expressed no Kv1.3 but, when chronically stimulated and activated ( I ), exhibited intense Kv1.3 expression and colocalization with CD8. ( J ) Isotype controls using nonspecific rabbit primary antibody followed by usual secondary label and with primary rabbit anti-human and nonspecific labeled mouse anti-rabbit secondary antibody showed no background staining.

    Techniques Used: Confocal Microscopy, Mass Spectrometry, Staining, Derivative Assay, Expressing, Labeling

    21) Product Images from "Increased ACh-Associated Immunoreactivity in Autonomic Centers in PTZ Kindling Model of Epilepsy"

    Article Title: Increased ACh-Associated Immunoreactivity in Autonomic Centers in PTZ Kindling Model of Epilepsy

    Journal: Biomedicines

    doi: 10.3390/biomedicines8050113

    Immunoreactivity results of Kir3.1 channel and M2 receptors in the VN. In both groups, increased immunoreactivity in cervical VN was observed. For female groups;  A : Image of cervical VN of control groups,  B : Image of thoracic VN of control groups,  C : Image of cervical VN of PTZ-kindled epilepsy,  D : Image of thoracic VN of PTZ-kindled epilepsy. For male groups;  E : Image of cervical VN of control groups,  F : Image of thoracic VN of control groups,  G : Image of cervical VN of PTZ-kindled epilepsy,  H : Image of thoracic VN of PTZ-kindled epilepsy. Pictures were taken at a magnification of ×200. Scale bar: 50 μm ( A ). Fold change in immunoreactivity for M2 receptors and Kir3.1 channel in the VN ( B ). For cervical VN, in terms of the immunoreactivity of the M2 receptor, a 1.6-fold change in male and a 1.8-fold change in female rats compared to controls can be seen; for cervical VN, in terms of the immunoreactivity of Kir3.1, a 2.2-fold change in male and a 2.4-fold change in female rats compared to thoracic VN can be seen, *  p
    Figure Legend Snippet: Immunoreactivity results of Kir3.1 channel and M2 receptors in the VN. In both groups, increased immunoreactivity in cervical VN was observed. For female groups; A : Image of cervical VN of control groups, B : Image of thoracic VN of control groups, C : Image of cervical VN of PTZ-kindled epilepsy, D : Image of thoracic VN of PTZ-kindled epilepsy. For male groups; E : Image of cervical VN of control groups, F : Image of thoracic VN of control groups, G : Image of cervical VN of PTZ-kindled epilepsy, H : Image of thoracic VN of PTZ-kindled epilepsy. Pictures were taken at a magnification of ×200. Scale bar: 50 μm ( A ). Fold change in immunoreactivity for M2 receptors and Kir3.1 channel in the VN ( B ). For cervical VN, in terms of the immunoreactivity of the M2 receptor, a 1.6-fold change in male and a 1.8-fold change in female rats compared to controls can be seen; for cervical VN, in terms of the immunoreactivity of Kir3.1, a 2.2-fold change in male and a 2.4-fold change in female rats compared to thoracic VN can be seen, * p

    Techniques Used:

    Examples of immunoreactivity for ACh receptors and Kir3.1 channel within the brainstem. Immunoreactivity was significantly increased for both ACh-associated centers in the represented images, the result for male rats was prepared. For female ( A ) and male ( B ) in terms of immunoreactivity at pons; A : Image of M2 receptors for control groups, B : Image of M2 receptors for PTZ-kindled of epilepsy, C : Image of Kir3.1 channels for control groups, D : Image of Kir3.1 channels of PTZ-kindled epilepsy. Pictures were taken at a magnification of ×200. Scale bar: 50 μm. Fold change in immunoreactivity for M2 receptors and Kir3.1 channel within the brainstem ( C ). For male, PTZ-kindled rats, 3.8-fold change in the M2 receptor and 2.7-fold change in Kir3.1 channel were observed when compared to the controls. For female PTZ-kindled rats, there was a 2.1-fold change in M2 receptors and 2.2-fold change in Kir3.1 channel when compared to the controls, * p
    Figure Legend Snippet: Examples of immunoreactivity for ACh receptors and Kir3.1 channel within the brainstem. Immunoreactivity was significantly increased for both ACh-associated centers in the represented images, the result for male rats was prepared. For female ( A ) and male ( B ) in terms of immunoreactivity at pons; A : Image of M2 receptors for control groups, B : Image of M2 receptors for PTZ-kindled of epilepsy, C : Image of Kir3.1 channels for control groups, D : Image of Kir3.1 channels of PTZ-kindled epilepsy. Pictures were taken at a magnification of ×200. Scale bar: 50 μm. Fold change in immunoreactivity for M2 receptors and Kir3.1 channel within the brainstem ( C ). For male, PTZ-kindled rats, 3.8-fold change in the M2 receptor and 2.7-fold change in Kir3.1 channel were observed when compared to the controls. For female PTZ-kindled rats, there was a 2.1-fold change in M2 receptors and 2.2-fold change in Kir3.1 channel when compared to the controls, * p

    Techniques Used:

    22) 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

    23) 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

    24) Product Images from "Potassium secretion by voltage-gated potassium channel Kv1.3 in the rat kidney"

    Article Title: Potassium secretion by voltage-gated potassium channel Kv1.3 in the rat kidney

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00697.2009

    Kv1.3 expression in collecting duct intercalated cells from kidneys of HK-fed rats. Shown is detection of principal cells with Dolichus biflorus aggutinin coupled to fluorescein (DBA-F; A ; green), intercalated cells with goat-anti-B1-H-VATPase antibody ( D and G ; green), and Kv1.3 with an anti-Kv1.3 antibody ( B , E , and H ; red) in collecting ducts of HK-fed rats by indirect immunofluorescence confocal microscopy. Note that Kv1.3 is localized only to cells lacking DBA-F staining ( C ; merged) and in presumably intercalated cells expressing apical H + -VATPase ( F and I ; merged). Arrows point to apical Kv1.3-H + -VATPase colocalization (yellow). Bars = 25 μm.
    Figure Legend Snippet: Kv1.3 expression in collecting duct intercalated cells from kidneys of HK-fed rats. Shown is detection of principal cells with Dolichus biflorus aggutinin coupled to fluorescein (DBA-F; A ; green), intercalated cells with goat-anti-B1-H-VATPase antibody ( D and G ; green), and Kv1.3 with an anti-Kv1.3 antibody ( B , E , and H ; red) in collecting ducts of HK-fed rats by indirect immunofluorescence confocal microscopy. Note that Kv1.3 is localized only to cells lacking DBA-F staining ( C ; merged) and in presumably intercalated cells expressing apical H + -VATPase ( F and I ; merged). Arrows point to apical Kv1.3-H + -VATPase colocalization (yellow). Bars = 25 μm.

    Techniques Used: Expressing, Immunofluorescence, Confocal Microscopy, Staining

    Dietary K + intake has no effect on Kv1.3 channel transcript and protein expression. A : real-time PCR assays were performed to detect the relative abundance of Kv1.3 transcript in Cx, OM, and IM from rats fed control K + (CK) and high-K + (HK) diets. There was no significant difference in the Kv1.3 mRNA expression in any region between the CK and HK groups ( n = 6). B : representative Western blot showing Kv1.3 in crude membranes from Cx, OM, and IM from rats CK and HK diets. Crude protein (30 μg) was loaded on each lane. No differences were observed in Kv1.3 protein expression between the CK and HK groups.
    Figure Legend Snippet: Dietary K + intake has no effect on Kv1.3 channel transcript and protein expression. A : real-time PCR assays were performed to detect the relative abundance of Kv1.3 transcript in Cx, OM, and IM from rats fed control K + (CK) and high-K + (HK) diets. There was no significant difference in the Kv1.3 mRNA expression in any region between the CK and HK groups ( n = 6). B : representative Western blot showing Kv1.3 in crude membranes from Cx, OM, and IM from rats CK and HK diets. Crude protein (30 μg) was loaded on each lane. No differences were observed in Kv1.3 protein expression between the CK and HK groups.

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

    Effect of dietary K + intake on Kv1.3 expression in plasma membranes. A : representative immunoblot of Kv1.3 in plasma membranes from Cx, OM, and IM of rats fed CK and HK diets. Thirty micrograms of protein was loaded on each lane. B : significant relative increase was detected in the 3 regions of kidney studied in response to dietary K + loading, suggesting increased trafficking or stability of Kv1.3 protein to/on plasma membranes. Values are mean ± SE; n = 6/dietary group. * P
    Figure Legend Snippet: Effect of dietary K + intake on Kv1.3 expression in plasma membranes. A : representative immunoblot of Kv1.3 in plasma membranes from Cx, OM, and IM of rats fed CK and HK diets. Thirty micrograms of protein was loaded on each lane. B : significant relative increase was detected in the 3 regions of kidney studied in response to dietary K + loading, suggesting increased trafficking or stability of Kv1.3 protein to/on plasma membranes. Values are mean ± SE; n = 6/dietary group. * P

    Techniques Used: Expressing

    Immunohistochemistry of Kv1.3 in the kidney Cx in rats fed CK ( A ) and HK diets ( B ). Cortical collecting ducts (CCDs) are observed with positive cytoplasmic ( A ) and polarized ( B , arrows) immunoreactivity. Higher magnification shows a cytoplasmic distribution in all CCD cells of Kv1.3 in CK rats ( C ); the inset at the bottom left shows a negative control (preincubation of the anti-Kv1.3 antibody with the control peptide). The HK diet enhances apical expression of Kv1.3 in a few cells ( D , arrows). Bars = 50 μm.
    Figure Legend Snippet: Immunohistochemistry of Kv1.3 in the kidney Cx in rats fed CK ( A ) and HK diets ( B ). Cortical collecting ducts (CCDs) are observed with positive cytoplasmic ( A ) and polarized ( B , arrows) immunoreactivity. Higher magnification shows a cytoplasmic distribution in all CCD cells of Kv1.3 in CK rats ( C ); the inset at the bottom left shows a negative control (preincubation of the anti-Kv1.3 antibody with the control peptide). The HK diet enhances apical expression of Kv1.3 in a few cells ( D , arrows). Bars = 50 μm.

    Techniques Used: Immunohistochemistry, Negative Control, Expressing

    25) Product Images from "Expression of Kv1.3 potassium channels regulates density of cortical interneurons"

    Article Title: Expression of Kv1.3 potassium channels regulates density of cortical interneurons

    Journal: Developmental neurobiology

    doi: 10.1002/dneu.22105

    Different classes of cortical interneurons express Kv1.3 channels. Double immunofluorescence staining was used to determine the presence of Kv1.3 channels in the different types of interneurons tested. In every case interneurons are labeled with secondary
    Figure Legend Snippet: Different classes of cortical interneurons express Kv1.3 channels. Double immunofluorescence staining was used to determine the presence of Kv1.3 channels in the different types of interneurons tested. In every case interneurons are labeled with secondary

    Techniques Used: Double Immunofluorescence Staining, Labeling

    Deletion of Kv1.3 affects cortical interneuron numbers, densities and cortical volume. A. The mean number of cortical interneurons is always higher in controls than in Kv1.3 −/− except for PV in which the trend is reversed. Also, the difference
    Figure Legend Snippet: Deletion of Kv1.3 affects cortical interneuron numbers, densities and cortical volume. A. The mean number of cortical interneurons is always higher in controls than in Kv1.3 −/− except for PV in which the trend is reversed. Also, the difference

    Techniques Used:

    Deletion of Kv1.3 affects cortical interneuron numbers and densities
    Figure Legend Snippet: Deletion of Kv1.3 affects cortical interneuron numbers and densities

    Techniques Used:

    Expression of the transcription factor CDP (CCAAT displacement protein), a marker for layers II/III/IV, appears mostly absent in layers III/IV of Kv1.3−/− mice while expression of ER81 (layer 5 marker) and FoxP2 (layer 6 marker) appear
    Figure Legend Snippet: Expression of the transcription factor CDP (CCAAT displacement protein), a marker for layers II/III/IV, appears mostly absent in layers III/IV of Kv1.3−/− mice while expression of ER81 (layer 5 marker) and FoxP2 (layer 6 marker) appear

    Techniques Used: Expressing, Marker, Mouse Assay

    Deletion of Kv1.3 increases the number of PV interneurons in the cerebral cortex. The upper diagrams show actual samplings. The punctate marks inside of the cortex represent positively stained cells plotted inside counting boxes (200 μm ×
    Figure Legend Snippet: Deletion of Kv1.3 increases the number of PV interneurons in the cerebral cortex. The upper diagrams show actual samplings. The punctate marks inside of the cortex represent positively stained cells plotted inside counting boxes (200 μm ×

    Techniques Used: Staining

    Deletion of Kv1.3 decreases the number of SOM interneurons in the cerebral cortex. The upper diagrams show the actual samplings. The punctate marks inside of the cortex represent positively stained cells plotted inside counting boxes, which were the same
    Figure Legend Snippet: Deletion of Kv1.3 decreases the number of SOM interneurons in the cerebral cortex. The upper diagrams show the actual samplings. The punctate marks inside of the cortex represent positively stained cells plotted inside counting boxes, which were the same

    Techniques Used: Staining

    A. Kv1.3 stains the extracellular membrane of all cultured primary cortical cells (not permeabilized) that were visualized using the confocal microscope. Cell cultures were obtained at E14.5 and staining was done at E15.5. Kv1.3 is visualized with an
    Figure Legend Snippet: A. Kv1.3 stains the extracellular membrane of all cultured primary cortical cells (not permeabilized) that were visualized using the confocal microscope. Cell cultures were obtained at E14.5 and staining was done at E15.5. Kv1.3 is visualized with an

    Techniques Used: Cell Culture, Microscopy, Staining

    26) 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

    Kv currents of Xenopus laevis oocytes in two-electrode voltage clamp experiments. Notes: Effects of PLGA nanoparticles or SPIONs on Xenopus laevis oocytes expressing ( A ) Kv1.3 and ( B ) Kv7.1. The oocytes were incubated with 500 ng/mL PLGA nanoparticles or SPIONs for 4 hours. The black curve (n=24/22) indicates the untreated control oocytes, and the currents are normalized to this curve. The blue curve (n=21/28) indicates the treatment with PLGA nanoparticles, the red curve (n=21/17) indicates treatment with SPIONs, the green curve (n=12/14) represents control oocytes without RNA injection and nanoparticle treatment as an example for all uninjected oocytes with and without nanoparticle treatment. Abbreviations: PLGA, poly(lactic-co-glycolic acid); SPIONs, starch-coated superparamagnetic iron oxide nanoparticles.
    Figure Legend Snippet: Kv currents of Xenopus laevis oocytes in two-electrode voltage clamp experiments. Notes: Effects of PLGA nanoparticles or SPIONs on Xenopus laevis oocytes expressing ( A ) Kv1.3 and ( B ) Kv7.1. The oocytes were incubated with 500 ng/mL PLGA nanoparticles or SPIONs for 4 hours. The black curve (n=24/22) indicates the untreated control oocytes, and the currents are normalized to this curve. The blue curve (n=21/28) indicates the treatment with PLGA nanoparticles, the red curve (n=21/17) indicates treatment with SPIONs, the green curve (n=12/14) represents control oocytes without RNA injection and nanoparticle treatment as an example for all uninjected oocytes with and without nanoparticle treatment. Abbreviations: PLGA, poly(lactic-co-glycolic acid); SPIONs, starch-coated superparamagnetic iron oxide nanoparticles.

    Techniques Used: Expressing, Incubation, Injection

    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

    27) 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

    28) 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

    29) 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

    30) Product Images from "Potassium Channel Kv1.3 Is Highly Expressed by Microglia in Human Alzheimer’s Disease"

    Article Title: Potassium Channel Kv1.3 Is Highly Expressed by Microglia in Human Alzheimer’s Disease

    Journal: Journal of Alzheimer's disease : JAD

    doi: 10.3233/JAD-141704

    Kv1.3 positive cells are found around Aβ aggregates in AD brain. A, B) Kv1.3 positive cells (brown) aggregate around Aβ plaques (blue-black). Arrows indicate Kv1.3 positive cells (A: 10×, B: 20×). C, D) Iba1 positive microglia,
    Figure Legend Snippet: Kv1.3 positive cells are found around Aβ aggregates in AD brain. A, B) Kv1.3 positive cells (brown) aggregate around Aβ plaques (blue-black). Arrows indicate Kv1.3 positive cells (A: 10×, B: 20×). C, D) Iba1 positive microglia,

    Techniques Used:

    Iba1 and Kv1.3 expression is increased in AD brains as compared to control brains. Left: Representative 20× images demonstrating low Iba1 and minimal Kv1.3 staining in a control brain (top panel) compared to strong Iba1 positivity and robust Kv1.3
    Figure Legend Snippet: Iba1 and Kv1.3 expression is increased in AD brains as compared to control brains. Left: Representative 20× images demonstrating low Iba1 and minimal Kv1.3 staining in a control brain (top panel) compared to strong Iba1 positivity and robust Kv1.3

    Techniques Used: Expressing, Staining

    Kv1.3 expression in frontal cortex of AD and non-AD controls as measured by immunoblotting. Kv1.3 expression was higher in the membrane fraction of AD brains ( n = 4) as compared to non-AD controls ( n = 5).
    Figure Legend Snippet: Kv1.3 expression in frontal cortex of AD and non-AD controls as measured by immunoblotting. Kv1.3 expression was higher in the membrane fraction of AD brains ( n = 4) as compared to non-AD controls ( n = 5).

    Techniques Used: Expressing

    Patterns of Kv1.3 channel expression in AD brains. Two staining patterns were observed. A) Kv1.3 staining in AD brain: “glial pattern” (20×). B) Secondary antibody control (20×). C) Kv1.3 staining in AD brain: “plaque-like”
    Figure Legend Snippet: Patterns of Kv1.3 channel expression in AD brains. Two staining patterns were observed. A) Kv1.3 staining in AD brain: “glial pattern” (20×). B) Secondary antibody control (20×). C) Kv1.3 staining in AD brain: “plaque-like”

    Techniques Used: Expressing, Staining

    Specificity of Kv1.3 antibody. Pre-adsorption with Kv1.3 immunizing peptide (8µg/ml) abolishes glial (A) as well as plaque-patterns (B) of Kv1.3 staining in frontal cortical regions of AD brain tissue.
    Figure Legend Snippet: Specificity of Kv1.3 antibody. Pre-adsorption with Kv1.3 immunizing peptide (8µg/ml) abolishes glial (A) as well as plaque-patterns (B) of Kv1.3 staining in frontal cortical regions of AD brain tissue.

    Techniques Used: Adsorption, Staining

    Kv1.3 channels in AD brains are predominantly expressed by Iba1 positive microglia. A) Kv1.3 (red) co-localizes robustly with Iba1 (green) [top panel] . B) Low levels of co-localization were observed between Kv1.3 (red) and GFAP (green) [bottom panel] .
    Figure Legend Snippet: Kv1.3 channels in AD brains are predominantly expressed by Iba1 positive microglia. A) Kv1.3 (red) co-localizes robustly with Iba1 (green) [top panel] . B) Low levels of co-localization were observed between Kv1.3 (red) and GFAP (green) [bottom panel] .

    Techniques Used:

    31) 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

    32) 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

    33) Product Images from "C5b-9-activated, Kv1.3 channels mediate oligodendrocyte cell cycle activation and dedifferentiation"

    Article Title: C5b-9-activated, Kv1.3 channels mediate oligodendrocyte cell cycle activation and dedifferentiation

    Journal: Experimental and molecular pathology

    doi: 10.1016/j.yexmp.2011.04.006

    NG2+  cells express Kv 1.3 and co-localized with C5b-9 in brains from MS patients
    Figure Legend Snippet: NG2+ cells express Kv 1.3 and co-localized with C5b-9 in brains from MS patients

    Techniques Used: Mass Spectrometry

    34) Product Images from "Overexpression of Delayed Rectifier K+ Channels Promotes In situ Proliferation of Leukocytes in Rat Kidneys with Advanced Chronic Renal Failure"

    Article Title: Overexpression of Delayed Rectifier K+ Channels Promotes In situ Proliferation of Leukocytes in Rat Kidneys with Advanced Chronic Renal Failure

    Journal: International Journal of Nephrology

    doi: 10.1155/2012/581581

    Kv1.3 and cell cycle marker expression in sham operated (sham) and advanced CRF rat kidneys. (A) Kv1.3 expression. (a) KCNA3 mRNA abundance in the renal cortex of sham operated (sham) and advanced CRF (CRF) rat kidneys. (b) and (c) Immunohistochemistry using antibody for Kv1.3 (brown) in sham operated and advanced CRF rat kidneys. High-power views of cortical interstitium. Magnification, ×60. (B) Cell cycle marker expression. The mRNA abundance of cyclin-dependent kinase 4 (Cdk4) (left) and p21 (right) in the renal cortex of sham operated and advanced CRF rat kidneys. # P
    Figure Legend Snippet: Kv1.3 and cell cycle marker expression in sham operated (sham) and advanced CRF rat kidneys. (A) Kv1.3 expression. (a) KCNA3 mRNA abundance in the renal cortex of sham operated (sham) and advanced CRF (CRF) rat kidneys. (b) and (c) Immunohistochemistry using antibody for Kv1.3 (brown) in sham operated and advanced CRF rat kidneys. High-power views of cortical interstitium. Magnification, ×60. (B) Cell cycle marker expression. The mRNA abundance of cyclin-dependent kinase 4 (Cdk4) (left) and p21 (right) in the renal cortex of sham operated and advanced CRF rat kidneys. # P

    Techniques Used: Marker, Expressing, Immunohistochemistry

    35) Product Images from "Granzyme B-Induced Neurotoxicity Is Mediated via Activation of PAR-1 Receptor and Kv1.3 Channel"

    Article Title: Granzyme B-Induced Neurotoxicity Is Mediated via Activation of PAR-1 Receptor and Kv1.3 Channel

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043950

    Immunostaining for Kv1.3 in autopsy brain tissue in patients with multiple sclerosis. (A) Section presents an area in the cortex showing a demyelinating lesion using hematoxylin and eosin staining. (B) Several neurons are seen within this lesion immunostaining for Kv1.3. (C) At higher magnification, cell body and neurites are immunostained for Kv1.3. (D) Staining with primary antibody only shows absence of immunostaining.
    Figure Legend Snippet: Immunostaining for Kv1.3 in autopsy brain tissue in patients with multiple sclerosis. (A) Section presents an area in the cortex showing a demyelinating lesion using hematoxylin and eosin staining. (B) Several neurons are seen within this lesion immunostaining for Kv1.3. (C) At higher magnification, cell body and neurites are immunostained for Kv1.3. (D) Staining with primary antibody only shows absence of immunostaining.

    Techniques Used: Immunostaining, Staining

    Schematic flow chart depicting possible mechanisms involved in GrB-mediated neuronal damage. Activated T cells release GrB into the supernatants. GrB induces neurotoxicity by binding and cleaving PAR-1 receptor. Activation of Gi-coupled PAR-1 then results in decreased cAMP levels, leading to Kv1.3 activation. Activation of Kv1.3 results in mitochondrial dysfunction and caspase-3 activation, which may lead to apoptosis. Kv1.3 activation can also increase Notch-1 activation, resulting in neurite damage.
    Figure Legend Snippet: Schematic flow chart depicting possible mechanisms involved in GrB-mediated neuronal damage. Activated T cells release GrB into the supernatants. GrB induces neurotoxicity by binding and cleaving PAR-1 receptor. Activation of Gi-coupled PAR-1 then results in decreased cAMP levels, leading to Kv1.3 activation. Activation of Kv1.3 results in mitochondrial dysfunction and caspase-3 activation, which may lead to apoptosis. Kv1.3 activation can also increase Notch-1 activation, resulting in neurite damage.

    Techniques Used: Flow Cytometry, Binding Assay, Activation Assay

    GrB activates Kv1.3 channel in neurons. (A) Human fetal neurons were treated with GrB (4 nM) for 24 hr. Cells were then fixed and immunostained for Kv1.3 and beta-III tubulin and analyzed by confocal microscopy. Representative photomicrographs from three independent experiments with similar results are shown. (B) Human fetal neurons were pretreated with cycloheximide (CHX, 100 µg/ml) or actinomycin D (Act D, 10 µM) for 30 min prior to GrB (4 nM) treatment. 24 hr later, cells were fixed and immunostained for Kv1.3 and analyzed by confocal microscopy. Representative photomicrographs from three independent experiments with similar results are shown. (C) Primary human neuronal cultures were first transfected with siRNA specific to Kv1.3 (KvSi). After 48 hr, GrB (4 nM) was used to treat the cells. Cells were fixed after 24 hr and immunostained for beta-III-tubulin. Neurite lengths were measured as detailed in Methods. Results represent average ± SEM from three independent experiments. (D) Human neuronal cells were transfected with PAR-1 specific siRNA (PARsi) or a nonspecific control siRNA (Nsi) 48 hr prior to GrB treatment and Western-blot analysis was used to detect Kv1.3 expression after 24 hr of GrB treatment. Representative blot is shown (Lane 1: control; lane 2: PARsi; lane 3: Nsi; lane 4: GrB; lane 5: GrB/PARsi: Lane 6: GrB/Nsi) and results are presented as average ± SEM from three independent experiments. (E) Primary human neuronal cultures were pretreated with corresponding inhibitors 30 min prior to GrB treatment (4 nM). Cell viability was determined using Cytoquantiblue assay 24 hr later. Results represent mean ± SEM. (F) Cells were incubated with a K free solution containing 5 uM PBFI AM for 2 hours. After washing, the cells were treated with GrB (10 nM) with/without MgTX (10 nM) pretreatment. Intracellular K+ concentration was determined by measuring the florescence at Ex 340 nM and Em 500 nM. Data represents mean ± SEM from five replicates.
    Figure Legend Snippet: GrB activates Kv1.3 channel in neurons. (A) Human fetal neurons were treated with GrB (4 nM) for 24 hr. Cells were then fixed and immunostained for Kv1.3 and beta-III tubulin and analyzed by confocal microscopy. Representative photomicrographs from three independent experiments with similar results are shown. (B) Human fetal neurons were pretreated with cycloheximide (CHX, 100 µg/ml) or actinomycin D (Act D, 10 µM) for 30 min prior to GrB (4 nM) treatment. 24 hr later, cells were fixed and immunostained for Kv1.3 and analyzed by confocal microscopy. Representative photomicrographs from three independent experiments with similar results are shown. (C) Primary human neuronal cultures were first transfected with siRNA specific to Kv1.3 (KvSi). After 48 hr, GrB (4 nM) was used to treat the cells. Cells were fixed after 24 hr and immunostained for beta-III-tubulin. Neurite lengths were measured as detailed in Methods. Results represent average ± SEM from three independent experiments. (D) Human neuronal cells were transfected with PAR-1 specific siRNA (PARsi) or a nonspecific control siRNA (Nsi) 48 hr prior to GrB treatment and Western-blot analysis was used to detect Kv1.3 expression after 24 hr of GrB treatment. Representative blot is shown (Lane 1: control; lane 2: PARsi; lane 3: Nsi; lane 4: GrB; lane 5: GrB/PARsi: Lane 6: GrB/Nsi) and results are presented as average ± SEM from three independent experiments. (E) Primary human neuronal cultures were pretreated with corresponding inhibitors 30 min prior to GrB treatment (4 nM). Cell viability was determined using Cytoquantiblue assay 24 hr later. Results represent mean ± SEM. (F) Cells were incubated with a K free solution containing 5 uM PBFI AM for 2 hours. After washing, the cells were treated with GrB (10 nM) with/without MgTX (10 nM) pretreatment. Intracellular K+ concentration was determined by measuring the florescence at Ex 340 nM and Em 500 nM. Data represents mean ± SEM from five replicates.

    Techniques Used: Confocal Microscopy, Activated Clotting Time Assay, Transfection, Western Blot, Expressing, Incubation, Concentration Assay

    Kv1.3 activation is specific to GrB-mediated neurotoxicity. A. Human neuronal cultures were treated with GrB (4 nM) and mitochondrial inhibitor 3NP (3 mM) with/without 30 min of pretreatment of MgTX (10 nM) or fluconazole (FCZ, 10 µM). After 24 hr, cells were collected and fixed. Duplicate coverslips from each treatment were immunostained for active caspase-3 and Kv1.3. Positive cells in each of nine predesigned fields were counted. Average of positive cells in each of the fields was calculated. Results represent average ± SEM from three independent experiments. B. Human neuronal cultures were treated with 6-OH-dopamine (50–200 µM) with/without 30 min of pretreatment of MgTX (10 nM) or rTrtyustoxin (10 nM). After 24 hr, cell viability was measured using Cellquanti-blue assay.
    Figure Legend Snippet: Kv1.3 activation is specific to GrB-mediated neurotoxicity. A. Human neuronal cultures were treated with GrB (4 nM) and mitochondrial inhibitor 3NP (3 mM) with/without 30 min of pretreatment of MgTX (10 nM) or fluconazole (FCZ, 10 µM). After 24 hr, cells were collected and fixed. Duplicate coverslips from each treatment were immunostained for active caspase-3 and Kv1.3. Positive cells in each of nine predesigned fields were counted. Average of positive cells in each of the fields was calculated. Results represent average ± SEM from three independent experiments. B. Human neuronal cultures were treated with 6-OH-dopamine (50–200 µM) with/without 30 min of pretreatment of MgTX (10 nM) or rTrtyustoxin (10 nM). After 24 hr, cell viability was measured using Cellquanti-blue assay.

    Techniques Used: Activation Assay

    Notch-1 pathway activation by GrB is mediated by PAR-1 and Kv1.3. Confluent HEK293 cells were transfected with wild 8XCBF-1 luciferase construct (w) or mutant control construct (m). (A) Cells were co-transfected with PAR-1 specific siRNA (PARSi) or negative siRNA (NSi) to determine the effect of PAR-1 on Notch-1 activation. After 48 hr, cells were treated with GrB (4 nM). Cell lysates were collected 48 hr later and luciferase activity was quantified. Results are average ± SEM from three independent experiments. (B) In another set of experiments, 48 hr after transfection, cells were treated with GrB (4 nM) with or without 30 min pretreatment with PTX (100 ng/ml) or MgTX (100 nM). Luciferase activity was quantified after an additional 48 hr. Results represent average ± SEM from six independent experiments.
    Figure Legend Snippet: Notch-1 pathway activation by GrB is mediated by PAR-1 and Kv1.3. Confluent HEK293 cells were transfected with wild 8XCBF-1 luciferase construct (w) or mutant control construct (m). (A) Cells were co-transfected with PAR-1 specific siRNA (PARSi) or negative siRNA (NSi) to determine the effect of PAR-1 on Notch-1 activation. After 48 hr, cells were treated with GrB (4 nM). Cell lysates were collected 48 hr later and luciferase activity was quantified. Results are average ± SEM from three independent experiments. (B) In another set of experiments, 48 hr after transfection, cells were treated with GrB (4 nM) with or without 30 min pretreatment with PTX (100 ng/ml) or MgTX (100 nM). Luciferase activity was quantified after an additional 48 hr. Results represent average ± SEM from six independent experiments.

    Techniques Used: Activation Assay, Transfection, Luciferase, Construct, Mutagenesis, Activity Assay

    36) 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

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Alomone Labs anti kv1 3
    Fyn modulates the posttranslational modification of <t>Kv1.3.</t> (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
    Anti Kv1 3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kv1 3/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv1 3 - by Bioz Stars, 2022-05
    95/100 stars
      Buy from Supplier

    Image Search Results


    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

    Journal: The Journal of Clinical Investigation

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

    doi: 10.1172/JCI136174

    Figure Lengend 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

    Article Snippet: The primary antibodies included anti-Kv1.3 (Alomone Labs, 1:1000) (Research Resource Identifier [RRID]: AB_2040151), anti-Kv1.3 (MilliporeSigma 1:1000) (RRID: AB_2265087), anti–p-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_331641), anti-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_330713), anti–p-Kv1.3 (MilliporeSigma, 1:1000, catalog SAB4504254), anti-PKCδ (Santa Cruz Biotechnology, 1:500) (RRID: AB_628145), anti-NLRP3 (AdipoGen, 1:1000) (RRID: AB_2490202), and anti–active MAPK (Promega, 1:2000).

    Techniques: 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

    Journal: The Journal of Clinical Investigation

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

    doi: 10.1172/JCI136174

    Figure Lengend 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

    Article Snippet: The primary antibodies included anti-Kv1.3 (Alomone Labs, 1:1000) (Research Resource Identifier [RRID]: AB_2040151), anti-Kv1.3 (MilliporeSigma 1:1000) (RRID: AB_2265087), anti–p-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_331641), anti-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_330713), anti–p-Kv1.3 (MilliporeSigma, 1:1000, catalog SAB4504254), anti-PKCδ (Santa Cruz Biotechnology, 1:500) (RRID: AB_628145), anti-NLRP3 (AdipoGen, 1:1000) (RRID: AB_2490202), and anti–active MAPK (Promega, 1:2000).

    Techniques: 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

    Journal: The Journal of Clinical Investigation

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

    doi: 10.1172/JCI136174

    Figure Lengend 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

    Article Snippet: The primary antibodies included anti-Kv1.3 (Alomone Labs, 1:1000) (Research Resource Identifier [RRID]: AB_2040151), anti-Kv1.3 (MilliporeSigma 1:1000) (RRID: AB_2265087), anti–p-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_331641), anti-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_330713), anti–p-Kv1.3 (MilliporeSigma, 1:1000, catalog SAB4504254), anti-PKCδ (Santa Cruz Biotechnology, 1:500) (RRID: AB_628145), anti-NLRP3 (AdipoGen, 1:1000) (RRID: AB_2490202), and anti–active MAPK (Promega, 1:2000).

    Techniques: 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

    Journal: The Journal of Clinical Investigation

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

    doi: 10.1172/JCI136174

    Figure Lengend 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

    Article Snippet: The primary antibodies included anti-Kv1.3 (Alomone Labs, 1:1000) (Research Resource Identifier [RRID]: AB_2040151), anti-Kv1.3 (MilliporeSigma 1:1000) (RRID: AB_2265087), anti–p-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_331641), anti-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_330713), anti–p-Kv1.3 (MilliporeSigma, 1:1000, catalog SAB4504254), anti-PKCδ (Santa Cruz Biotechnology, 1:500) (RRID: AB_628145), anti-NLRP3 (AdipoGen, 1:1000) (RRID: AB_2490202), and anti–active MAPK (Promega, 1:2000).

    Techniques: 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

    Journal: The Journal of Clinical Investigation

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

    doi: 10.1172/JCI136174

    Figure Lengend 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

    Article Snippet: The primary antibodies included anti-Kv1.3 (Alomone Labs, 1:1000) (Research Resource Identifier [RRID]: AB_2040151), anti-Kv1.3 (MilliporeSigma 1:1000) (RRID: AB_2265087), anti–p-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_331641), anti-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_330713), anti–p-Kv1.3 (MilliporeSigma, 1:1000, catalog SAB4504254), anti-PKCδ (Santa Cruz Biotechnology, 1:500) (RRID: AB_628145), anti-NLRP3 (AdipoGen, 1:1000) (RRID: AB_2490202), and anti–active MAPK (Promega, 1:2000).

    Techniques: 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

    Journal: The Journal of Clinical Investigation

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

    doi: 10.1172/JCI136174

    Figure Lengend 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

    Article Snippet: The primary antibodies included anti-Kv1.3 (Alomone Labs, 1:1000) (Research Resource Identifier [RRID]: AB_2040151), anti-Kv1.3 (MilliporeSigma 1:1000) (RRID: AB_2265087), anti–p-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_331641), anti-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_330713), anti–p-Kv1.3 (MilliporeSigma, 1:1000, catalog SAB4504254), anti-PKCδ (Santa Cruz Biotechnology, 1:500) (RRID: AB_628145), anti-NLRP3 (AdipoGen, 1:1000) (RRID: AB_2490202), and anti–active MAPK (Promega, 1:2000).

    Techniques: 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

    Journal: The Journal of Clinical Investigation

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

    doi: 10.1172/JCI136174

    Figure Lengend 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

    Article Snippet: The primary antibodies included anti-Kv1.3 (Alomone Labs, 1:1000) (Research Resource Identifier [RRID]: AB_2040151), anti-Kv1.3 (MilliporeSigma 1:1000) (RRID: AB_2265087), anti–p-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_331641), anti-p38 (Cell Signaling Technology, 1:1000) (RRID: AB_330713), anti–p-Kv1.3 (MilliporeSigma, 1:1000, catalog SAB4504254), anti-PKCδ (Santa Cruz Biotechnology, 1:500) (RRID: AB_628145), anti-NLRP3 (AdipoGen, 1:1000) (RRID: AB_2490202), and anti–active MAPK (Promega, 1:2000).

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

    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

    Journal: Journal of Neurophysiology

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

    doi: 10.1152/jn.00494.2010

    Figure Lengend 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

    Article Snippet: The anti-Kv1.3 antibody (Alomone) recognized a prominent band with the apparent molecular mass of ∼65 kDa in both postnatal day 1 and 36 tissue ( ).

    Techniques: 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

    Journal: Journal of Neurophysiology

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

    doi: 10.1152/jn.00494.2010

    Figure Lengend 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

    Article Snippet: The anti-Kv1.3 antibody (Alomone) recognized a prominent band with the apparent molecular mass of ∼65 kDa in both postnatal day 1 and 36 tissue ( ).

    Techniques: 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

    Journal: Journal of Neurophysiology

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

    doi: 10.1152/jn.00494.2010

    Figure Lengend 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

    Article Snippet: The anti-Kv1.3 antibody (Alomone) recognized a prominent band with the apparent molecular mass of ∼65 kDa in both postnatal day 1 and 36 tissue ( ).

    Techniques: Labeling

    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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

    Figure Lengend 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

    Article Snippet: After incubation for 60 min in blocking solution (1% BSA, 20 mM Gly, 0.05% Triton X-100, PBS-K+), the cells were treated with a rabbit anti-Kv1.3 antibody (1/20, Alomone) in 1% BSA, 20 mM Gly, and 0.05% Triton X-100 in PBS-K+ and incubated for 90 min. After 3 washes, the preparations were incubated for 60 min with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:200; Molecular Probes), washed and mounted with Mowiol (Calbiochem).

    Techniques: 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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

    Figure Lengend 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

    Article Snippet: After incubation for 60 min in blocking solution (1% BSA, 20 mM Gly, 0.05% Triton X-100, PBS-K+), the cells were treated with a rabbit anti-Kv1.3 antibody (1/20, Alomone) in 1% BSA, 20 mM Gly, and 0.05% Triton X-100 in PBS-K+ and incubated for 90 min. After 3 washes, the preparations were incubated for 60 min with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:200; Molecular Probes), washed and mounted with Mowiol (Calbiochem).

    Techniques: 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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

    Figure Lengend 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

    Article Snippet: After incubation for 60 min in blocking solution (1% BSA, 20 mM Gly, 0.05% Triton X-100, PBS-K+), the cells were treated with a rabbit anti-Kv1.3 antibody (1/20, Alomone) in 1% BSA, 20 mM Gly, and 0.05% Triton X-100 in PBS-K+ and incubated for 90 min. After 3 washes, the preparations were incubated for 60 min with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:200; Molecular Probes), washed and mounted with Mowiol (Calbiochem).

    Techniques: 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.

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

    Figure Lengend 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.

    Article Snippet: After incubation for 60 min in blocking solution (1% BSA, 20 mM Gly, 0.05% Triton X-100, PBS-K+), the cells were treated with a rabbit anti-Kv1.3 antibody (1/20, Alomone) in 1% BSA, 20 mM Gly, and 0.05% Triton X-100 in PBS-K+ and incubated for 90 min. After 3 washes, the preparations were incubated for 60 min with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:200; Molecular Probes), washed and mounted with Mowiol (Calbiochem).

    Techniques:

    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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

    Figure Lengend 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

    Article Snippet: After incubation for 60 min in blocking solution (1% BSA, 20 mM Gly, 0.05% Triton X-100, PBS-K+), the cells were treated with a rabbit anti-Kv1.3 antibody (1/20, Alomone) in 1% BSA, 20 mM Gly, and 0.05% Triton X-100 in PBS-K+ and incubated for 90 min. After 3 washes, the preparations were incubated for 60 min with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:200; Molecular Probes), washed and mounted with Mowiol (Calbiochem).

    Techniques: 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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

    Figure Lengend 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

    Article Snippet: After incubation for 60 min in blocking solution (1% BSA, 20 mM Gly, 0.05% Triton X-100, PBS-K+), the cells were treated with a rabbit anti-Kv1.3 antibody (1/20, Alomone) in 1% BSA, 20 mM Gly, and 0.05% Triton X-100 in PBS-K+ and incubated for 90 min. After 3 washes, the preparations were incubated for 60 min with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:200; Molecular Probes), washed and mounted with Mowiol (Calbiochem).

    Techniques:

    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

    Journal: Glia

    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

    doi: 10.1002/glia.23847

    Figure Lengend 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

    Article Snippet: Validation of polyclonal rabbit anti‐Kv1.3 antibody.

    Techniques: 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

    Journal: Glia

    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

    doi: 10.1002/glia.23847

    Figure Lengend 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

    Article Snippet: Validation of polyclonal rabbit anti‐Kv1.3 antibody.

    Techniques: 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

    Journal: Glia

    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

    doi: 10.1002/glia.23847

    Figure Lengend 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

    Article Snippet: Validation of polyclonal rabbit anti‐Kv1.3 antibody.

    Techniques: 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

    Journal: Glia

    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

    doi: 10.1002/glia.23847

    Figure Lengend 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

    Article Snippet: Validation of polyclonal rabbit anti‐Kv1.3 antibody.

    Techniques: 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

    Journal: Glia

    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

    doi: 10.1002/glia.23847

    Figure Lengend 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

    Article Snippet: Validation of polyclonal rabbit anti‐Kv1.3 antibody.

    Techniques: 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

    Journal: Glia

    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

    doi: 10.1002/glia.23847

    Figure Lengend 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

    Article Snippet: Validation of polyclonal rabbit anti‐Kv1.3 antibody.

    Techniques: Inhibition