machr  (Alomone Labs)


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    Structured Review

    Alomone Labs machr
    Machr, 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/machr/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
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    machr - by Bioz Stars, 2022-12
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    Alomone Labs anti kv1 3 kcna3 antibody
    MSU effects on protein expression of K + channels and Hsp70, degradation of <t>Kv1.5</t> proteins and Kv1.5 channel currents in J774.1 cells. a IB analysis of Kv1.5 and <t>Kv1.3</t> proteins and Hsp70 in the cytosolic and membrane fraction of LPS-primed and MSU-stimulated cells and untreated cells. Na + /K + ATPase and β-actin were used as the plasma membrane and protein loading control, respectively. b Degradation of Kv1.5 proteins. The LPS-primed MSU-treated cells (LPS + MSU) or untreated cells (none) were chased for the indicated times after an addition of cycloheximide. Representative blots are shown. The densities of Kv1.5 was normalized to the density at time 0 and β-actin. Bar graph shows the half-life of the Kv1.5 proteins (n = 4). * p
    Anti Kv1 3 Kcna3 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kv1 3 kcna3 antibody/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv1 3 kcna3 antibody - by Bioz Stars, 2022-12
    95/100 stars
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    MSU effects on protein expression of K + channels and Hsp70, degradation of Kv1.5 proteins and Kv1.5 channel currents in J774.1 cells. a IB analysis of Kv1.5 and Kv1.3 proteins and Hsp70 in the cytosolic and membrane fraction of LPS-primed and MSU-stimulated cells and untreated cells. Na + /K + ATPase and β-actin were used as the plasma membrane and protein loading control, respectively. b Degradation of Kv1.5 proteins. The LPS-primed MSU-treated cells (LPS + MSU) or untreated cells (none) were chased for the indicated times after an addition of cycloheximide. Representative blots are shown. The densities of Kv1.5 was normalized to the density at time 0 and β-actin. Bar graph shows the half-life of the Kv1.5 proteins (n = 4). * p

    Journal: Molecular Biology Reports

    Article Title: Kv1.5 channel mediates monosodium urate-induced activation of NLRP3 inflammasome in macrophages and arrhythmogenic effects of urate on cardiomyocytes

    doi: 10.1007/s11033-022-07378-1

    Figure Lengend Snippet: MSU effects on protein expression of K + channels and Hsp70, degradation of Kv1.5 proteins and Kv1.5 channel currents in J774.1 cells. a IB analysis of Kv1.5 and Kv1.3 proteins and Hsp70 in the cytosolic and membrane fraction of LPS-primed and MSU-stimulated cells and untreated cells. Na + /K + ATPase and β-actin were used as the plasma membrane and protein loading control, respectively. b Degradation of Kv1.5 proteins. The LPS-primed MSU-treated cells (LPS + MSU) or untreated cells (none) were chased for the indicated times after an addition of cycloheximide. Representative blots are shown. The densities of Kv1.5 was normalized to the density at time 0 and β-actin. Bar graph shows the half-life of the Kv1.5 proteins (n = 4). * p

    Article Snippet: The antibodies used were as follows: NLRP3 (Cell Signaling, Cat# 15101), ASC (AdipoGen, Cat# AG-25B-0006) and caspase-1 (AdipoGen, Cat# AG-20B-0042, AG-20B-0048), IL-1β (R & D systems, Cat# AF-401-NA), Kv1.5 and Kv1.3 (alomone Labs, Cat# APC-004, APC-002), Na+ /K+ ATPase α-1 (upstate, Cat# 05-369), Hsp70 (Stressgen, Cat# SPA-810), and β-actin (Calbiochem, Cat# CP01).

    Techniques: Expressing

    Effects of Kv1.5 on intracellular K + concentration, ASC oligomerization and speck formation in J774.1 cells. Intracellular K + concentrations of LPS-primed cells stimulated with MSU in either the presence or absence of DPO-1 (1 μM) or the selective Kv1.3 channel blocker PAP-1 (50 nM) ( a ), and those with the introduction of a siRNA against Kv1.5 or Kv1.3 or a scramble siRNA 24 h before the LPS and MSU treatments ( b ) (n = 5–11). * p

    Journal: Molecular Biology Reports

    Article Title: Kv1.5 channel mediates monosodium urate-induced activation of NLRP3 inflammasome in macrophages and arrhythmogenic effects of urate on cardiomyocytes

    doi: 10.1007/s11033-022-07378-1

    Figure Lengend Snippet: Effects of Kv1.5 on intracellular K + concentration, ASC oligomerization and speck formation in J774.1 cells. Intracellular K + concentrations of LPS-primed cells stimulated with MSU in either the presence or absence of DPO-1 (1 μM) or the selective Kv1.3 channel blocker PAP-1 (50 nM) ( a ), and those with the introduction of a siRNA against Kv1.5 or Kv1.3 or a scramble siRNA 24 h before the LPS and MSU treatments ( b ) (n = 5–11). * p

    Article Snippet: The antibodies used were as follows: NLRP3 (Cell Signaling, Cat# 15101), ASC (AdipoGen, Cat# AG-25B-0006) and caspase-1 (AdipoGen, Cat# AG-20B-0042, AG-20B-0048), IL-1β (R & D systems, Cat# AF-401-NA), Kv1.5 and Kv1.3 (alomone Labs, Cat# APC-004, APC-002), Na+ /K+ ATPase α-1 (upstate, Cat# 05-369), Hsp70 (Stressgen, Cat# SPA-810), and β-actin (Calbiochem, Cat# CP01).

    Techniques: Concentration Assay

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1016/j.jbc.2021.101497

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

    Article Snippet: MgTx and primary antibodies against Kv1.3 were from Alomone Labs, p38, p-p38, ERK, p-ERK, JNK, p-JNK, Histone H3, NF-кB p65, NFATc1, GAPDH, and horseradish peroxidase–conjugated secondary antibodies were from Cell Signaling Technologies.

    Techniques: Expressing, Inhibition

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1016/j.jbc.2021.101497

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

    Article Snippet: MgTx and primary antibodies against Kv1.3 were from Alomone Labs, p38, p-p38, ERK, p-ERK, JNK, p-JNK, Histone H3, NF-кB p65, NFATc1, GAPDH, and horseradish peroxidase–conjugated secondary antibodies were from Cell Signaling Technologies.

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

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1016/j.jbc.2021.101497

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

    Article Snippet: MgTx and primary antibodies against Kv1.3 were from Alomone Labs, p38, p-p38, ERK, p-ERK, JNK, p-JNK, Histone H3, NF-кB p65, NFATc1, GAPDH, and horseradish peroxidase–conjugated secondary antibodies were from Cell Signaling Technologies.

    Techniques: Concentration Assay, Activation Assay

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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

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

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

    Techniques: Fractionation, Marker, Labeling

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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

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

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

    Techniques: Flow Cytometry

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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

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

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

    Techniques: Incubation, Cell Cycle Assay

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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

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

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

    Techniques:

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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

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

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

    Techniques: Immunofluorescence, Infection, shRNA

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

    Journal: Cancers

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

    doi: 10.3390/cancers13174457

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

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

    Techniques:

    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: Cells were labeled with anti-Kv1.3-FITC (Alomone, 1/50), anti-CD3 Alexa 647-conjugated antibody and anti-CD19 Alexa 488-conjugated antibody (BioLegend, 1/80).

    Techniques: Marker, Staining

    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

    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

    Article Snippet: Cells were labeled with anti-Kv1.3-FITC (Alomone, 1/50), anti-CD3 Alexa 647-conjugated antibody and anti-CD19 Alexa 488-conjugated antibody (BioLegend, 1/80).

    Techniques: Expressing, Imaging, Transfection, Staining, Flow Cytometry

    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

    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

    Article Snippet: Cells were labeled with anti-Kv1.3-FITC (Alomone, 1/50), anti-CD3 Alexa 647-conjugated antibody and anti-CD19 Alexa 488-conjugated antibody (BioLegend, 1/80).

    Techniques: Activation Assay, Incubation, Staining, Whole Genome Amplification, Marker, Immunolabeling

    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

    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

    Article Snippet: Cells were labeled with anti-Kv1.3-FITC (Alomone, 1/50), anti-CD3 Alexa 647-conjugated antibody and anti-CD19 Alexa 488-conjugated antibody (BioLegend, 1/80).

    Techniques: Over Expression, Transfection, Expressing, Cell Culture

    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: Cells were labeled with anti-Kv1.3-FITC (Alomone, 1/50), anti-CD3 Alexa 647-conjugated antibody and anti-CD19 Alexa 488-conjugated antibody (BioLegend, 1/80).

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

    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

    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

    Article Snippet: Cells were labeled with anti-Kv1.3-FITC (Alomone, 1/50), anti-CD3 Alexa 647-conjugated antibody and anti-CD19 Alexa 488-conjugated antibody (BioLegend, 1/80).

    Techniques: Activation Assay, Expressing, Staining