anti kv1 3 extracellular  (Alomone Labs)


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    Structured Review

    Alomone Labs anti kv1 3 extracellular
    <t>Kv1.3</t> and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.
    Anti Kv1 3 Extracellular, 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 extracellular/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv1 3 extracellular - by Bioz Stars, 2023-02
    93/100 stars

    Images

    1) Product Images from "KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology"

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-94015-9

    Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.
    Figure Legend Snippet: Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.

    Techniques Used: Expressing, Negative Control, Western Blot, Immunoprecipitation, Isolation

    KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p < 0.05 (Student’s t -test). Kv1.3CFP-Kv1.3YFP was used as a positive control, whereas Kv1.3CFP-KCNE2YFP served as the negative control. ( G , H ) Representative current traces obtained from control Jurkat T cells ( G ) and cells positively transfected with KCNE4CFP ( H ). Cells were clamped at -60 mV, and K + currents elicited by 250 ms voltage steps from -80 mV to +80 mV in 10-mV increments. ( I ) I/Imax plotted against voltage (mV). Black circles, control cells (n = 21); white circles, YFP (n = 4); gray circles, + KCNE4CFP cells (n = 6). ( J ) Kv currents recorded in the whole-cell configuration by depolarizing ramps from -100 to +100 mV. Each black trace represents the average ± SE (shadowed in gray) of several ramps. Traces shown for control Jurkat T cells (n = 22) and + KCNE4CFP (n = 5).
    Figure Legend Snippet: KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p < 0.05 (Student’s t -test). Kv1.3CFP-Kv1.3YFP was used as a positive control, whereas Kv1.3CFP-KCNE2YFP served as the negative control. ( G , H ) Representative current traces obtained from control Jurkat T cells ( G ) and cells positively transfected with KCNE4CFP ( H ). Cells were clamped at -60 mV, and K + currents elicited by 250 ms voltage steps from -80 mV to +80 mV in 10-mV increments. ( I ) I/Imax plotted against voltage (mV). Black circles, control cells (n = 21); white circles, YFP (n = 4); gray circles, + KCNE4CFP cells (n = 6). ( J ) Kv currents recorded in the whole-cell configuration by depolarizing ramps from -100 to +100 mV. Each black trace represents the average ± SE (shadowed in gray) of several ramps. Traces shown for control Jurkat T cells (n = 22) and + KCNE4CFP (n = 5).

    Techniques Used: Expressing, Imaging, Transfection, Staining, Flow Cytometry, Positive Control, Negative Control

    KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p < 0.05, vs. control; n = 5–7 independent experiments (Student’s t -test). ( C ) Cell cycle analysis with Hoechst 33342 Jurkat T cells. Cell cycle distribution (% of cells in each phase) after 24 h in the presence of FBS. G 0 /G 1 -phase, dark gray bars; S-phase, white bars; G 2 /M-phase of the cell cycle, light gray bars. Mean ± SE (n = 3 independent experiments). ( D ) Relative cell size of Jurkat cells measured in a Countess™ automated cell counter. The size of control cells corresponded to a diameter of 10.9 ± 0.6 µm. Values are the mean ± SE from n = 11 independent experiments (*p < 0.05, + KCNE4 vs. control; Student’s t -test). ( E ) KCNE4CFP overexpression hampered IL-2 production in activated Jurkat T lymphocytes. Cells were cultured in the presence or absence of PMA (80 nM) and PHA (5 mg/ml) for 24 and 48 h. IL-2 production (ng/ml) from basal and PMA/PHA-stimulated cells was measured. Values are mean ± SE of n = 5–8 different experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA and Tukey’s post hoc test). ( F – I ) The presence of KCNE4CFP increased apoptosis in Jurkat T cells. ( F ) Representative results of the apoptosis assay (Annexin V-APC—DAPI assay) in control Jurkat cells. ( G ) Apoptosis experiments in KCNE4CFP-positive Jurkat cells. ( H ) Percentage of control Jurkat T cells transfected with YFP and KCNE4CFP under different conditions. ( I ) Percentage of apoptotic control, YFP and KCNE4CFP cells. Values are the mean ± SE from n = 4–6 independent experiments; **p < 0.01; ***p < 0.001, Student’s t -test). Panels ( B , D , E and G ) White bars, control Jurkat T-cells; black bars, YFP-transfected cells; gray bars, KCNE4CFP-positive cells.
    Figure Legend Snippet: KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p < 0.05, vs. control; n = 5–7 independent experiments (Student’s t -test). ( C ) Cell cycle analysis with Hoechst 33342 Jurkat T cells. Cell cycle distribution (% of cells in each phase) after 24 h in the presence of FBS. G 0 /G 1 -phase, dark gray bars; S-phase, white bars; G 2 /M-phase of the cell cycle, light gray bars. Mean ± SE (n = 3 independent experiments). ( D ) Relative cell size of Jurkat cells measured in a Countess™ automated cell counter. The size of control cells corresponded to a diameter of 10.9 ± 0.6 µm. Values are the mean ± SE from n = 11 independent experiments (*p < 0.05, + KCNE4 vs. control; Student’s t -test). ( E ) KCNE4CFP overexpression hampered IL-2 production in activated Jurkat T lymphocytes. Cells were cultured in the presence or absence of PMA (80 nM) and PHA (5 mg/ml) for 24 and 48 h. IL-2 production (ng/ml) from basal and PMA/PHA-stimulated cells was measured. Values are mean ± SE of n = 5–8 different experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA and Tukey’s post hoc test). ( F – I ) The presence of KCNE4CFP increased apoptosis in Jurkat T cells. ( F ) Representative results of the apoptosis assay (Annexin V-APC—DAPI assay) in control Jurkat cells. ( G ) Apoptosis experiments in KCNE4CFP-positive Jurkat cells. ( H ) Percentage of control Jurkat T cells transfected with YFP and KCNE4CFP under different conditions. ( I ) Percentage of apoptotic control, YFP and KCNE4CFP cells. Values are the mean ± SE from n = 4–6 independent experiments; **p < 0.01; ***p < 0.001, Student’s t -test). Panels ( B , D , E and G ) White bars, control Jurkat T-cells; black bars, YFP-transfected cells; gray bars, KCNE4CFP-positive cells.

    Techniques Used: Over Expression, Transfection, Expressing, Cell Culture, Cell Cycle Assay, Apoptosis Assay

    KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.
    Figure Legend Snippet: KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.

    Techniques Used: Marker, Staining

    LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p < 0.05 vs 0 h, Student’s t test. ( D ) Representative western blot of Kv1.5, Kv1.3 and KCNE4. The expression of iNOS monitored cellular activation in the presence of LPS. β-actin was used as a loading control. Representative cropped blots, clearly delineated by vertical white lines, are shown. ( E ) Relative expression of protein abundance. Kv1.3, black circles; Kv1.5, gray circles; KCNE4, white circles. ( F ) Coimmunoprecipitation of Kv1.3 and KCNE4 in CY15 cells in the absence (control) or presence of LPS for 24 h. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and KCNE4 (IP: KCNE4) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. IP−: Immunoprecipitated in absence of antibodies. Representative cropped blots from SM, clearly delineated by vertical black lines, are shown. (G) Relative coimmunoprecipitation of KCNE4 with Kv1.3 (Kv1.3/KCNE4 ratio) and Kv1.3 with KCNE4 (KCNE4/Kv1.3 ratio). Black bars, Kv1.3 was immunoprecipitated (IP: Kv1.3), and the associated KCNE4 (CoIP: KCNE4) was analyzed. White bars, KCNE4 was immunoprecipitated (IP: KCNE4), and the association of Kv1.3 (CoIP: Kv1.3) was analyzed. (H) The Kv1.3/KCNE4 ratio calculated from the protein expression of Kv1.3 and KCNE4 in LPS-treated CY15 dendritic cells. Values are mean ± SE of n = 4 independent samples. **p < 0.01 One-way ANOVA with post hoc Tukey’s test.
    Figure Legend Snippet: LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p < 0.05 vs 0 h, Student’s t test. ( D ) Representative western blot of Kv1.5, Kv1.3 and KCNE4. The expression of iNOS monitored cellular activation in the presence of LPS. β-actin was used as a loading control. Representative cropped blots, clearly delineated by vertical white lines, are shown. ( E ) Relative expression of protein abundance. Kv1.3, black circles; Kv1.5, gray circles; KCNE4, white circles. ( F ) Coimmunoprecipitation of Kv1.3 and KCNE4 in CY15 cells in the absence (control) or presence of LPS for 24 h. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and KCNE4 (IP: KCNE4) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. IP−: Immunoprecipitated in absence of antibodies. Representative cropped blots from SM, clearly delineated by vertical black lines, are shown. (G) Relative coimmunoprecipitation of KCNE4 with Kv1.3 (Kv1.3/KCNE4 ratio) and Kv1.3 with KCNE4 (KCNE4/Kv1.3 ratio). Black bars, Kv1.3 was immunoprecipitated (IP: Kv1.3), and the associated KCNE4 (CoIP: KCNE4) was analyzed. White bars, KCNE4 was immunoprecipitated (IP: KCNE4), and the association of Kv1.3 (CoIP: Kv1.3) was analyzed. (H) The Kv1.3/KCNE4 ratio calculated from the protein expression of Kv1.3 and KCNE4 in LPS-treated CY15 dendritic cells. Values are mean ± SE of n = 4 independent samples. **p < 0.01 One-way ANOVA with post hoc Tukey’s test.

    Techniques Used: Activation Assay, Expressing, Staining, Western Blot, Immunoprecipitation

    LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p < 0.01 vs control (Student’s t-test).
    Figure Legend Snippet: LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p < 0.01 vs control (Student’s t-test).

    Techniques Used: Activation Assay, Incubation, Staining, Marker, Immunolabeling

    anti kv1 3 extracellular  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
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  • 93

    Structured Review

    Alomone Labs anti kv1 3 extracellular
    <t>Kv1.3</t> and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.
    Anti Kv1 3 Extracellular, 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 extracellular/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv1 3 extracellular - by Bioz Stars, 2023-02
    93/100 stars

    Images

    1) Product Images from "KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology"

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-94015-9

    Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.
    Figure Legend Snippet: Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.

    Techniques Used: Expressing, Negative Control, Western Blot, Immunoprecipitation, Isolation

    KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p < 0.05 (Student’s t -test). Kv1.3CFP-Kv1.3YFP was used as a positive control, whereas Kv1.3CFP-KCNE2YFP served as the negative control. ( G , H ) Representative current traces obtained from control Jurkat T cells ( G ) and cells positively transfected with KCNE4CFP ( H ). Cells were clamped at -60 mV, and K + currents elicited by 250 ms voltage steps from -80 mV to +80 mV in 10-mV increments. ( I ) I/Imax plotted against voltage (mV). Black circles, control cells (n = 21); white circles, YFP (n = 4); gray circles, + KCNE4CFP cells (n = 6). ( J ) Kv currents recorded in the whole-cell configuration by depolarizing ramps from -100 to +100 mV. Each black trace represents the average ± SE (shadowed in gray) of several ramps. Traces shown for control Jurkat T cells (n = 22) and + KCNE4CFP (n = 5).
    Figure Legend Snippet: KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p < 0.05 (Student’s t -test). Kv1.3CFP-Kv1.3YFP was used as a positive control, whereas Kv1.3CFP-KCNE2YFP served as the negative control. ( G , H ) Representative current traces obtained from control Jurkat T cells ( G ) and cells positively transfected with KCNE4CFP ( H ). Cells were clamped at -60 mV, and K + currents elicited by 250 ms voltage steps from -80 mV to +80 mV in 10-mV increments. ( I ) I/Imax plotted against voltage (mV). Black circles, control cells (n = 21); white circles, YFP (n = 4); gray circles, + KCNE4CFP cells (n = 6). ( J ) Kv currents recorded in the whole-cell configuration by depolarizing ramps from -100 to +100 mV. Each black trace represents the average ± SE (shadowed in gray) of several ramps. Traces shown for control Jurkat T cells (n = 22) and + KCNE4CFP (n = 5).

    Techniques Used: Expressing, Imaging, Transfection, Staining, Flow Cytometry, Positive Control, Negative Control

    KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p < 0.05, vs. control; n = 5–7 independent experiments (Student’s t -test). ( C ) Cell cycle analysis with Hoechst 33342 Jurkat T cells. Cell cycle distribution (% of cells in each phase) after 24 h in the presence of FBS. G 0 /G 1 -phase, dark gray bars; S-phase, white bars; G 2 /M-phase of the cell cycle, light gray bars. Mean ± SE (n = 3 independent experiments). ( D ) Relative cell size of Jurkat cells measured in a Countess™ automated cell counter. The size of control cells corresponded to a diameter of 10.9 ± 0.6 µm. Values are the mean ± SE from n = 11 independent experiments (*p < 0.05, + KCNE4 vs. control; Student’s t -test). ( E ) KCNE4CFP overexpression hampered IL-2 production in activated Jurkat T lymphocytes. Cells were cultured in the presence or absence of PMA (80 nM) and PHA (5 mg/ml) for 24 and 48 h. IL-2 production (ng/ml) from basal and PMA/PHA-stimulated cells was measured. Values are mean ± SE of n = 5–8 different experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA and Tukey’s post hoc test). ( F – I ) The presence of KCNE4CFP increased apoptosis in Jurkat T cells. ( F ) Representative results of the apoptosis assay (Annexin V-APC—DAPI assay) in control Jurkat cells. ( G ) Apoptosis experiments in KCNE4CFP-positive Jurkat cells. ( H ) Percentage of control Jurkat T cells transfected with YFP and KCNE4CFP under different conditions. ( I ) Percentage of apoptotic control, YFP and KCNE4CFP cells. Values are the mean ± SE from n = 4–6 independent experiments; **p < 0.01; ***p < 0.001, Student’s t -test). Panels ( B , D , E and G ) White bars, control Jurkat T-cells; black bars, YFP-transfected cells; gray bars, KCNE4CFP-positive cells.
    Figure Legend Snippet: KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p < 0.05, vs. control; n = 5–7 independent experiments (Student’s t -test). ( C ) Cell cycle analysis with Hoechst 33342 Jurkat T cells. Cell cycle distribution (% of cells in each phase) after 24 h in the presence of FBS. G 0 /G 1 -phase, dark gray bars; S-phase, white bars; G 2 /M-phase of the cell cycle, light gray bars. Mean ± SE (n = 3 independent experiments). ( D ) Relative cell size of Jurkat cells measured in a Countess™ automated cell counter. The size of control cells corresponded to a diameter of 10.9 ± 0.6 µm. Values are the mean ± SE from n = 11 independent experiments (*p < 0.05, + KCNE4 vs. control; Student’s t -test). ( E ) KCNE4CFP overexpression hampered IL-2 production in activated Jurkat T lymphocytes. Cells were cultured in the presence or absence of PMA (80 nM) and PHA (5 mg/ml) for 24 and 48 h. IL-2 production (ng/ml) from basal and PMA/PHA-stimulated cells was measured. Values are mean ± SE of n = 5–8 different experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA and Tukey’s post hoc test). ( F – I ) The presence of KCNE4CFP increased apoptosis in Jurkat T cells. ( F ) Representative results of the apoptosis assay (Annexin V-APC—DAPI assay) in control Jurkat cells. ( G ) Apoptosis experiments in KCNE4CFP-positive Jurkat cells. ( H ) Percentage of control Jurkat T cells transfected with YFP and KCNE4CFP under different conditions. ( I ) Percentage of apoptotic control, YFP and KCNE4CFP cells. Values are the mean ± SE from n = 4–6 independent experiments; **p < 0.01; ***p < 0.001, Student’s t -test). Panels ( B , D , E and G ) White bars, control Jurkat T-cells; black bars, YFP-transfected cells; gray bars, KCNE4CFP-positive cells.

    Techniques Used: Over Expression, Transfection, Expressing, Cell Culture, Cell Cycle Assay, Apoptosis Assay

    KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.
    Figure Legend Snippet: KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.

    Techniques Used: Marker, Staining

    LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p < 0.05 vs 0 h, Student’s t test. ( D ) Representative western blot of Kv1.5, Kv1.3 and KCNE4. The expression of iNOS monitored cellular activation in the presence of LPS. β-actin was used as a loading control. Representative cropped blots, clearly delineated by vertical white lines, are shown. ( E ) Relative expression of protein abundance. Kv1.3, black circles; Kv1.5, gray circles; KCNE4, white circles. ( F ) Coimmunoprecipitation of Kv1.3 and KCNE4 in CY15 cells in the absence (control) or presence of LPS for 24 h. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and KCNE4 (IP: KCNE4) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. IP−: Immunoprecipitated in absence of antibodies. Representative cropped blots from SM, clearly delineated by vertical black lines, are shown. (G) Relative coimmunoprecipitation of KCNE4 with Kv1.3 (Kv1.3/KCNE4 ratio) and Kv1.3 with KCNE4 (KCNE4/Kv1.3 ratio). Black bars, Kv1.3 was immunoprecipitated (IP: Kv1.3), and the associated KCNE4 (CoIP: KCNE4) was analyzed. White bars, KCNE4 was immunoprecipitated (IP: KCNE4), and the association of Kv1.3 (CoIP: Kv1.3) was analyzed. (H) The Kv1.3/KCNE4 ratio calculated from the protein expression of Kv1.3 and KCNE4 in LPS-treated CY15 dendritic cells. Values are mean ± SE of n = 4 independent samples. **p < 0.01 One-way ANOVA with post hoc Tukey’s test.
    Figure Legend Snippet: LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p < 0.05 vs 0 h, Student’s t test. ( D ) Representative western blot of Kv1.5, Kv1.3 and KCNE4. The expression of iNOS monitored cellular activation in the presence of LPS. β-actin was used as a loading control. Representative cropped blots, clearly delineated by vertical white lines, are shown. ( E ) Relative expression of protein abundance. Kv1.3, black circles; Kv1.5, gray circles; KCNE4, white circles. ( F ) Coimmunoprecipitation of Kv1.3 and KCNE4 in CY15 cells in the absence (control) or presence of LPS for 24 h. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and KCNE4 (IP: KCNE4) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. IP−: Immunoprecipitated in absence of antibodies. Representative cropped blots from SM, clearly delineated by vertical black lines, are shown. (G) Relative coimmunoprecipitation of KCNE4 with Kv1.3 (Kv1.3/KCNE4 ratio) and Kv1.3 with KCNE4 (KCNE4/Kv1.3 ratio). Black bars, Kv1.3 was immunoprecipitated (IP: Kv1.3), and the associated KCNE4 (CoIP: KCNE4) was analyzed. White bars, KCNE4 was immunoprecipitated (IP: KCNE4), and the association of Kv1.3 (CoIP: Kv1.3) was analyzed. (H) The Kv1.3/KCNE4 ratio calculated from the protein expression of Kv1.3 and KCNE4 in LPS-treated CY15 dendritic cells. Values are mean ± SE of n = 4 independent samples. **p < 0.01 One-way ANOVA with post hoc Tukey’s test.

    Techniques Used: Activation Assay, Expressing, Staining, Western Blot, Immunoprecipitation

    LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p < 0.01 vs control (Student’s t-test).
    Figure Legend Snippet: LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p < 0.01 vs control (Student’s t-test).

    Techniques Used: Activation Assay, Incubation, Staining, Marker, Immunolabeling

    guinea pig anti k v 1 3 extracellular antibody  (Alomone Labs)


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    Alomone Labs guinea pig anti k v 1 3 extracellular antibody
    Guinea Pig Anti K V 1 3 Extracellular 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
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    polyclonal guinea pig anti ano1  (Alomone Labs)


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    Alomone Labs polyclonal guinea pig anti ano1
    Polyclonal Guinea Pig Anti Ano1, 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
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    kv 1 3 extracellular antibody  (Alomone Labs)


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    Alomone Labs kv 1 3 extracellular antibody
    Kv 1 3 Extracellular 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
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    anti kv1 3 polyclonal serum  (Alomone Labs)


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    Alomone Labs anti kv1 3 polyclonal serum
    (A) <t>Kv1.3</t> gene expression was detected using real-time PCR in NPC cultures treated with GrB (4 nM) for 1-3 hours. GADPH was used as an internal control. Dose-dependent increase in Kv1.3 mRNA expression was noted. Data represent mean ± SEM from four independent experiments. (B) NPC cultures were treated with GrB (4nM) for 24 hours and immunostained with antibodies to Kv1.3 and nestin (Red: nestin, Green: Kv1.3). (C) NPC cultures were treated with GrB (1-4 nM) for 24 hours and the cell lysates were collected for detecting Kv1.3 production using western-blot analysis. This shows a dose-dependent increase in Kv1.3 detection.
    Anti Kv1 3 Polyclonal Serum, 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
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    1) Product Images from "Activated T cells Inhibit Neurogenesis by Releasing Granzyme B: Rescue by Kv1.3 blockers"

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

    Journal:

    doi: 10.1523/JNEUROSCI.0311-10.2010

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

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

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

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

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

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

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

    Techniques Used: Real-time Polymerase Chain Reaction

    anti kv1 3 polyclonal serum  (Alomone Labs)


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    Alomone Labs anti kv1 3 polyclonal serum
    Anti Kv1 3 Polyclonal Serum, 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
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    guinea pig gp intestine  (Alomone Labs)


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    Alomone Labs anti kv1 3 extracellular
    <t>Kv1.3</t> and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.
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    <t>Kv1.3</t> and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.
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    Alomone Labs polyclonal guinea pig anti ano1
    <t>Kv1.3</t> and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.
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    Alomone Labs kv 1 3 extracellular antibody
    <t>Kv1.3</t> and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.
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    Alomone Labs anti kv1 3 polyclonal serum
    (A) <t>Kv1.3</t> gene expression was detected using real-time PCR in NPC cultures treated with GrB (4 nM) for 1-3 hours. GADPH was used as an internal control. Dose-dependent increase in Kv1.3 mRNA expression was noted. Data represent mean ± SEM from four independent experiments. (B) NPC cultures were treated with GrB (4nM) for 24 hours and immunostained with antibodies to Kv1.3 and nestin (Red: nestin, Green: Kv1.3). (C) NPC cultures were treated with GrB (1-4 nM) for 24 hours and the cell lysates were collected for detecting Kv1.3 production using western-blot analysis. This shows a dose-dependent increase in Kv1.3 detection.
    Anti Kv1 3 Polyclonal Serum, 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
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    Alomone Labs guinea pig gp intestine
    (A) <t>Kv1.3</t> gene expression was detected using real-time PCR in NPC cultures treated with GrB (4 nM) for 1-3 hours. GADPH was used as an internal control. Dose-dependent increase in Kv1.3 mRNA expression was noted. Data represent mean ± SEM from four independent experiments. (B) NPC cultures were treated with GrB (4nM) for 24 hours and immunostained with antibodies to Kv1.3 and nestin (Red: nestin, Green: Kv1.3). (C) NPC cultures were treated with GrB (1-4 nM) for 24 hours and the cell lysates were collected for detecting Kv1.3 production using western-blot analysis. This shows a dose-dependent increase in Kv1.3 detection.
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    Image Search Results


    Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.

    Article Snippet: Next, the cells were incubated with anti-KCNE4 (1/50, Proteintech) or anti-Kv1.3 extracellular (1/150, Alomone) antibodies with 10% goat serum in 0.05% Triton X-100 PBS-K + for 1 h 45 min at RT.

    Techniques: Expressing, Negative Control, Western Blot, Immunoprecipitation, Isolation

    KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p < 0.05 (Student’s t -test). Kv1.3CFP-Kv1.3YFP was used as a positive control, whereas Kv1.3CFP-KCNE2YFP served as the negative control. ( G , H ) Representative current traces obtained from control Jurkat T cells ( G ) and cells positively transfected with KCNE4CFP ( H ). Cells were clamped at -60 mV, and K + currents elicited by 250 ms voltage steps from -80 mV to +80 mV in 10-mV increments. ( I ) I/Imax plotted against voltage (mV). Black circles, control cells (n = 21); white circles, YFP (n = 4); gray circles, + KCNE4CFP cells (n = 6). ( J ) Kv currents recorded in the whole-cell configuration by depolarizing ramps from -100 to +100 mV. Each black trace represents the average ± SE (shadowed in gray) of several ramps. Traces shown for control Jurkat T cells (n = 22) and + KCNE4CFP (n = 5).

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p < 0.05 (Student’s t -test). Kv1.3CFP-Kv1.3YFP was used as a positive control, whereas Kv1.3CFP-KCNE2YFP served as the negative control. ( G , H ) Representative current traces obtained from control Jurkat T cells ( G ) and cells positively transfected with KCNE4CFP ( H ). Cells were clamped at -60 mV, and K + currents elicited by 250 ms voltage steps from -80 mV to +80 mV in 10-mV increments. ( I ) I/Imax plotted against voltage (mV). Black circles, control cells (n = 21); white circles, YFP (n = 4); gray circles, + KCNE4CFP cells (n = 6). ( J ) Kv currents recorded in the whole-cell configuration by depolarizing ramps from -100 to +100 mV. Each black trace represents the average ± SE (shadowed in gray) of several ramps. Traces shown for control Jurkat T cells (n = 22) and + KCNE4CFP (n = 5).

    Article Snippet: Next, the cells were incubated with anti-KCNE4 (1/50, Proteintech) or anti-Kv1.3 extracellular (1/150, Alomone) antibodies with 10% goat serum in 0.05% Triton X-100 PBS-K + for 1 h 45 min at RT.

    Techniques: Expressing, Imaging, Transfection, Staining, Flow Cytometry, Positive Control, Negative Control

    KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p < 0.05, vs. control; n = 5–7 independent experiments (Student’s t -test). ( C ) Cell cycle analysis with Hoechst 33342 Jurkat T cells. Cell cycle distribution (% of cells in each phase) after 24 h in the presence of FBS. G 0 /G 1 -phase, dark gray bars; S-phase, white bars; G 2 /M-phase of the cell cycle, light gray bars. Mean ± SE (n = 3 independent experiments). ( D ) Relative cell size of Jurkat cells measured in a Countess™ automated cell counter. The size of control cells corresponded to a diameter of 10.9 ± 0.6 µm. Values are the mean ± SE from n = 11 independent experiments (*p < 0.05, + KCNE4 vs. control; Student’s t -test). ( E ) KCNE4CFP overexpression hampered IL-2 production in activated Jurkat T lymphocytes. Cells were cultured in the presence or absence of PMA (80 nM) and PHA (5 mg/ml) for 24 and 48 h. IL-2 production (ng/ml) from basal and PMA/PHA-stimulated cells was measured. Values are mean ± SE of n = 5–8 different experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA and Tukey’s post hoc test). ( F – I ) The presence of KCNE4CFP increased apoptosis in Jurkat T cells. ( F ) Representative results of the apoptosis assay (Annexin V-APC—DAPI assay) in control Jurkat cells. ( G ) Apoptosis experiments in KCNE4CFP-positive Jurkat cells. ( H ) Percentage of control Jurkat T cells transfected with YFP and KCNE4CFP under different conditions. ( I ) Percentage of apoptotic control, YFP and KCNE4CFP cells. Values are the mean ± SE from n = 4–6 independent experiments; **p < 0.01; ***p < 0.001, Student’s t -test). Panels ( B , D , E and G ) White bars, control Jurkat T-cells; black bars, YFP-transfected cells; gray bars, KCNE4CFP-positive cells.

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p < 0.05, vs. control; n = 5–7 independent experiments (Student’s t -test). ( C ) Cell cycle analysis with Hoechst 33342 Jurkat T cells. Cell cycle distribution (% of cells in each phase) after 24 h in the presence of FBS. G 0 /G 1 -phase, dark gray bars; S-phase, white bars; G 2 /M-phase of the cell cycle, light gray bars. Mean ± SE (n = 3 independent experiments). ( D ) Relative cell size of Jurkat cells measured in a Countess™ automated cell counter. The size of control cells corresponded to a diameter of 10.9 ± 0.6 µm. Values are the mean ± SE from n = 11 independent experiments (*p < 0.05, + KCNE4 vs. control; Student’s t -test). ( E ) KCNE4CFP overexpression hampered IL-2 production in activated Jurkat T lymphocytes. Cells were cultured in the presence or absence of PMA (80 nM) and PHA (5 mg/ml) for 24 and 48 h. IL-2 production (ng/ml) from basal and PMA/PHA-stimulated cells was measured. Values are mean ± SE of n = 5–8 different experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA and Tukey’s post hoc test). ( F – I ) The presence of KCNE4CFP increased apoptosis in Jurkat T cells. ( F ) Representative results of the apoptosis assay (Annexin V-APC—DAPI assay) in control Jurkat cells. ( G ) Apoptosis experiments in KCNE4CFP-positive Jurkat cells. ( H ) Percentage of control Jurkat T cells transfected with YFP and KCNE4CFP under different conditions. ( I ) Percentage of apoptotic control, YFP and KCNE4CFP cells. Values are the mean ± SE from n = 4–6 independent experiments; **p < 0.01; ***p < 0.001, Student’s t -test). Panels ( B , D , E and G ) White bars, control Jurkat T-cells; black bars, YFP-transfected cells; gray bars, KCNE4CFP-positive cells.

    Article Snippet: Next, the cells were incubated with anti-KCNE4 (1/50, Proteintech) or anti-Kv1.3 extracellular (1/150, Alomone) antibodies with 10% goat serum in 0.05% Triton X-100 PBS-K + for 1 h 45 min at RT.

    Techniques: Over Expression, Transfection, Expressing, Cell Culture, Cell Cycle Assay, Apoptosis Assay

    KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.

    Article Snippet: Next, the cells were incubated with anti-KCNE4 (1/50, Proteintech) or anti-Kv1.3 extracellular (1/150, Alomone) antibodies with 10% goat serum in 0.05% Triton X-100 PBS-K + for 1 h 45 min at RT.

    Techniques: Marker, Staining

    LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p < 0.05 vs 0 h, Student’s t test. ( D ) Representative western blot of Kv1.5, Kv1.3 and KCNE4. The expression of iNOS monitored cellular activation in the presence of LPS. β-actin was used as a loading control. Representative cropped blots, clearly delineated by vertical white lines, are shown. ( E ) Relative expression of protein abundance. Kv1.3, black circles; Kv1.5, gray circles; KCNE4, white circles. ( F ) Coimmunoprecipitation of Kv1.3 and KCNE4 in CY15 cells in the absence (control) or presence of LPS for 24 h. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and KCNE4 (IP: KCNE4) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. IP−: Immunoprecipitated in absence of antibodies. Representative cropped blots from SM, clearly delineated by vertical black lines, are shown. (G) Relative coimmunoprecipitation of KCNE4 with Kv1.3 (Kv1.3/KCNE4 ratio) and Kv1.3 with KCNE4 (KCNE4/Kv1.3 ratio). Black bars, Kv1.3 was immunoprecipitated (IP: Kv1.3), and the associated KCNE4 (CoIP: KCNE4) was analyzed. White bars, KCNE4 was immunoprecipitated (IP: KCNE4), and the association of Kv1.3 (CoIP: Kv1.3) was analyzed. (H) The Kv1.3/KCNE4 ratio calculated from the protein expression of Kv1.3 and KCNE4 in LPS-treated CY15 dendritic cells. Values are mean ± SE of n = 4 independent samples. **p < 0.01 One-way ANOVA with post hoc Tukey’s test.

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p < 0.05 vs 0 h, Student’s t test. ( D ) Representative western blot of Kv1.5, Kv1.3 and KCNE4. The expression of iNOS monitored cellular activation in the presence of LPS. β-actin was used as a loading control. Representative cropped blots, clearly delineated by vertical white lines, are shown. ( E ) Relative expression of protein abundance. Kv1.3, black circles; Kv1.5, gray circles; KCNE4, white circles. ( F ) Coimmunoprecipitation of Kv1.3 and KCNE4 in CY15 cells in the absence (control) or presence of LPS for 24 h. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and KCNE4 (IP: KCNE4) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. IP−: Immunoprecipitated in absence of antibodies. Representative cropped blots from SM, clearly delineated by vertical black lines, are shown. (G) Relative coimmunoprecipitation of KCNE4 with Kv1.3 (Kv1.3/KCNE4 ratio) and Kv1.3 with KCNE4 (KCNE4/Kv1.3 ratio). Black bars, Kv1.3 was immunoprecipitated (IP: Kv1.3), and the associated KCNE4 (CoIP: KCNE4) was analyzed. White bars, KCNE4 was immunoprecipitated (IP: KCNE4), and the association of Kv1.3 (CoIP: Kv1.3) was analyzed. (H) The Kv1.3/KCNE4 ratio calculated from the protein expression of Kv1.3 and KCNE4 in LPS-treated CY15 dendritic cells. Values are mean ± SE of n = 4 independent samples. **p < 0.01 One-way ANOVA with post hoc Tukey’s test.

    Article Snippet: Next, the cells were incubated with anti-KCNE4 (1/50, Proteintech) or anti-Kv1.3 extracellular (1/150, Alomone) antibodies with 10% goat serum in 0.05% Triton X-100 PBS-K + for 1 h 45 min at RT.

    Techniques: Activation Assay, Expressing, Staining, Western Blot, Immunoprecipitation

    LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p < 0.01 vs control (Student’s t-test).

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p < 0.01 vs control (Student’s t-test).

    Article Snippet: Next, the cells were incubated with anti-KCNE4 (1/50, Proteintech) or anti-Kv1.3 extracellular (1/150, Alomone) antibodies with 10% goat serum in 0.05% Triton X-100 PBS-K + for 1 h 45 min at RT.

    Techniques: Activation Assay, Incubation, Staining, Marker, Immunolabeling

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

    Journal:

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

    doi: 10.1523/JNEUROSCI.0311-10.2010

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

    Article Snippet: Kv1.3 expression was also monitored in NPC cells by immunocytochemistry and western blot using anti-Kv1.3 polyclonal serum (1: 100, Alomone Labs, Jerusalem, Israel).

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

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

    Journal:

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

    doi: 10.1523/JNEUROSCI.0311-10.2010

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

    Article Snippet: Kv1.3 expression was also monitored in NPC cells by immunocytochemistry and western blot using anti-Kv1.3 polyclonal serum (1: 100, Alomone Labs, Jerusalem, Israel).

    Techniques: Cell Culture, Transfection, Concentration Assay, Immunostaining

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

    Journal:

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

    doi: 10.1523/JNEUROSCI.0311-10.2010

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

    Article Snippet: Kv1.3 expression was also monitored in NPC cells by immunocytochemistry and western blot using anti-Kv1.3 polyclonal serum (1: 100, Alomone Labs, Jerusalem, Israel).

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

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

    Journal:

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

    doi: 10.1523/JNEUROSCI.0311-10.2010

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

    Article Snippet: Kv1.3 expression was also monitored in NPC cells by immunocytochemistry and western blot using anti-Kv1.3 polyclonal serum (1: 100, Alomone Labs, Jerusalem, Israel).

    Techniques: Real-time Polymerase Chain Reaction