anti kv1 3 rabbit polyclonal  (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
  • 93

    Structured Review

    Alomone Labs anti kv1 3 rabbit polyclonal
    Expression of the potassium channel <t>Kv1.3.</t> Protein extracts were prepared from control and iron-fed C8B4 microglia. Western blot analysis was carried out to determine the level of Kv1.3 in the microglia. Bands for the protein were observed in both control and iron-fed microglia. Levels of tubulin were also determined to verify protein loading. The results showed a significant ( p
    Anti Kv1 3 Rabbit Polyclonal, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kv1 3 rabbit polyclonal/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv1 3 rabbit polyclonal - by Bioz Stars, 2022-07
    93/100 stars

    Images

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

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

    Journal: Biomolecules

    doi: 10.3390/biom8030067

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

    Techniques Used: Expressing, Western Blot

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Alomone Labs rabbit polyclonal anti kv1 3 antibody
    <t>Kv1.3</t> channels are recruited at the interface between CD3/CD28 beads and T cells
    Rabbit Polyclonal Anti Kv1 3 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti kv1 3 antibody/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti kv1 3 antibody - by Bioz Stars, 2022-07
    93/100 stars
      Buy from Supplier

    Image Search Results


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

    Journal:

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

    doi:

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

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

    Techniques:

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

    Journal:

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

    doi:

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

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

    Techniques:

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

    Journal:

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

    doi:

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

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

    Techniques:

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

    Journal:

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

    doi:

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

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

    Techniques:

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

    Journal:

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

    doi:

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

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

    Techniques: Activation Assay

    Differential kinetics of Kv1.3 channel reorganization in the IS

    Journal:

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

    doi:

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

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

    Techniques:

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

    Journal:

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

    doi:

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

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

    Techniques:

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

    Journal:

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

    doi:

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

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

    Techniques:

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

    Journal: Biomolecules

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

    doi: 10.3390/biom8030067

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

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

    Techniques: Expressing, Western Blot

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

    Journal: The Journal of Physiology

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

    doi: 10.1113/jphysiol.2002.017376

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

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

    Techniques: Western Blot, Transfection, SDS Page

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

    Journal: The Journal of Physiology

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

    doi: 10.1113/jphysiol.2002.017376

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

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

    Techniques: Immunoprecipitation

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

    Journal: The Journal of Physiology

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

    doi: 10.1113/jphysiol.2002.017376

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

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

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

    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