mouse anti kca3 1  (Alomone Labs)


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    Alomone Labs mouse anti kca3 1
    Role of TRPV4 and <t>KCa3.1</t> in alteration of membrane potential and Ca 2+ entry in astrocytes following OGD. a–f Changes in membrane potential in response to activation of KCa3.1 channels and TRPV4 channels in astrocytes exposed to OGD 1 h. a , b 1-EBIO was added to WT astrocytes and membrane potential measured with or without OGD or HC 067047. Data are presented as means ± SEM. n = 10–20. *** p
    Mouse Anti Kca3 1, 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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    mouse anti kca3 1 - by Bioz Stars, 2022-01
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    1) Product Images from "The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke"

    Article Title: The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-017-0973-8

    Role of TRPV4 and KCa3.1 in alteration of membrane potential and Ca 2+ entry in astrocytes following OGD. a–f Changes in membrane potential in response to activation of KCa3.1 channels and TRPV4 channels in astrocytes exposed to OGD 1 h. a , b 1-EBIO was added to WT astrocytes and membrane potential measured with or without OGD or HC 067047. Data are presented as means ± SEM. n = 10–20. *** p
    Figure Legend Snippet: Role of TRPV4 and KCa3.1 in alteration of membrane potential and Ca 2+ entry in astrocytes following OGD. a–f Changes in membrane potential in response to activation of KCa3.1 channels and TRPV4 channels in astrocytes exposed to OGD 1 h. a , b 1-EBIO was added to WT astrocytes and membrane potential measured with or without OGD or HC 067047. Data are presented as means ± SEM. n = 10–20. *** p

    Techniques Used: Activation Assay

    KCa3.1 and TRPV4 co-localized in primary cultured astrocytes and mouse brain cortex. Double immunofluorescence images of KCa3.1 (green) and TRPV4 (red) in normal mouse brains ( a , b ), and primary cultured astrocytes ( c , d ). Note the strong co-localization indicated by merge yellow fluorescence, quantification of the co-localization observed in experiments as shown in g. ( e ) The histograms represent the ratio of the mean Pearson correlation coefficient calculated from the co-labeling in a number of samples, as indicated above the bar. Scale bar: 25 μm
    Figure Legend Snippet: KCa3.1 and TRPV4 co-localized in primary cultured astrocytes and mouse brain cortex. Double immunofluorescence images of KCa3.1 (green) and TRPV4 (red) in normal mouse brains ( a , b ), and primary cultured astrocytes ( c , d ). Note the strong co-localization indicated by merge yellow fluorescence, quantification of the co-localization observed in experiments as shown in g. ( e ) The histograms represent the ratio of the mean Pearson correlation coefficient calculated from the co-labeling in a number of samples, as indicated above the bar. Scale bar: 25 μm

    Techniques Used: Cell Culture, Immunofluorescence, Fluorescence, Labeling

    Involvement of KCa3.1 in OGD-induced reactive astrogliosis. a , b Representative western blot showing GFAP expression in cultured astrocytes treated with OGD for 4 h in the presence of 1 μM TRAM-34 and 10 μM HC 067047. Quantification of western blot for GFAP expression ( n = 3). Data are presented as means ± SEM. # p
    Figure Legend Snippet: Involvement of KCa3.1 in OGD-induced reactive astrogliosis. a , b Representative western blot showing GFAP expression in cultured astrocytes treated with OGD for 4 h in the presence of 1 μM TRAM-34 and 10 μM HC 067047. Quantification of western blot for GFAP expression ( n = 3). Data are presented as means ± SEM. # p

    Techniques Used: Western Blot, Expressing, Cell Culture

    Upregulation of KCa3.1 channels and GFAP in mouse brains following pMCAO. a , b Western blot analysis of lysates from 10-week-old male WT mice following 1, 3, 6, or 12 h of pMCAO analyzed by antibodies to KCa3.1 ( a ) and GFAP ( b ). Data represent the means ± SEM of KCa3.1 and GFAP density normalized to β-actin values for n = 3. * p
    Figure Legend Snippet: Upregulation of KCa3.1 channels and GFAP in mouse brains following pMCAO. a , b Western blot analysis of lysates from 10-week-old male WT mice following 1, 3, 6, or 12 h of pMCAO analyzed by antibodies to KCa3.1 ( a ) and GFAP ( b ). Data represent the means ± SEM of KCa3.1 and GFAP density normalized to β-actin values for n = 3. * p

    Techniques Used: Western Blot, Mouse Assay

    KCa3.1 deficiency reduces infarction volume and improves of neurological conditions. Focal cerebral ischemia was induced by pMCAO. a , c , and e Representative TTC staining of five corresponding coronal brain sections of a 10-week-old male WT mouse and a 10 week-old male KCa3.1 −/− mouse after 3 h ( a ), 6 h ( c ), and 24 h ( e ) of pMCAO. b , d , and f . Quantitative analysis of infarction volume in a, c, and e, respectively. Data are presented as means ± SEM. n = 6. * p
    Figure Legend Snippet: KCa3.1 deficiency reduces infarction volume and improves of neurological conditions. Focal cerebral ischemia was induced by pMCAO. a , c , and e Representative TTC staining of five corresponding coronal brain sections of a 10-week-old male WT mouse and a 10 week-old male KCa3.1 −/− mouse after 3 h ( a ), 6 h ( c ), and 24 h ( e ) of pMCAO. b , d , and f . Quantitative analysis of infarction volume in a, c, and e, respectively. Data are presented as means ± SEM. n = 6. * p

    Techniques Used: Staining

    Upregulation of KCa3.1, GFAP, and TRPV4 channels following OGD in cultured astrocytes. Western blot analysis of ( a ) KCa3.1, ( b ) GFAP, and ( c ) TRPV4 expression after OGD-treatment for 0, 1, 3, 4, 6, 12 h. Data represent the means ± SEM of KCa3.1, GFAP, and TRPV4 density normalized to β-actin values for n = 3 cultures. * p
    Figure Legend Snippet: Upregulation of KCa3.1, GFAP, and TRPV4 channels following OGD in cultured astrocytes. Western blot analysis of ( a ) KCa3.1, ( b ) GFAP, and ( c ) TRPV4 expression after OGD-treatment for 0, 1, 3, 4, 6, 12 h. Data represent the means ± SEM of KCa3.1, GFAP, and TRPV4 density normalized to β-actin values for n = 3 cultures. * p

    Techniques Used: Cell Culture, Western Blot, Expressing

    Decreased glial activation and neuronal loss in brains of KCa3.1 deletion mice following pMCAO. Reactive astrocytes ( a ), activated microglia ( b ), and neurons ( c ) from the hippocampal CA1 regions of WT or KCa3.1 −/− mice at 6 h after pMCAO were visualized by GFAP, Iba1, and NeuN immunostaining, respectively. At least four coronal slices from each mouse brain and at least three brains of each genotype were used for immunostaining and counting. n = 4 per group. Scale bar: 75 μm. Data represent means ± SEM. * p
    Figure Legend Snippet: Decreased glial activation and neuronal loss in brains of KCa3.1 deletion mice following pMCAO. Reactive astrocytes ( a ), activated microglia ( b ), and neurons ( c ) from the hippocampal CA1 regions of WT or KCa3.1 −/− mice at 6 h after pMCAO were visualized by GFAP, Iba1, and NeuN immunostaining, respectively. At least four coronal slices from each mouse brain and at least three brains of each genotype were used for immunostaining and counting. n = 4 per group. Scale bar: 75 μm. Data represent means ± SEM. * p

    Techniques Used: Activation Assay, Mouse Assay, Immunostaining

    2) Product Images from "The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease"

    Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-019-1682-2

    AKT modulation is crucial for KCa3.1-mediated ER stress in microglia. a , b Representative blots of p-AKT and total AKT in SNpc from a WT, WT+MPTP, KCa3.1 −/− , KCa3.1 −/− +MPTP group mice and from b control, MPTP, MPTP+Se, Se group mice. Data are presented as the mean ± SEM ( n = 3–5). Western blot was repeated three times and showed similar results. The OD value of p-AKT was normalized to that of AKT. * p
    Figure Legend Snippet: AKT modulation is crucial for KCa3.1-mediated ER stress in microglia. a , b Representative blots of p-AKT and total AKT in SNpc from a WT, WT+MPTP, KCa3.1 −/− , KCa3.1 −/− +MPTP group mice and from b control, MPTP, MPTP+Se, Se group mice. Data are presented as the mean ± SEM ( n = 3–5). Western blot was repeated three times and showed similar results. The OD value of p-AKT was normalized to that of AKT. * p

    Techniques Used: Mouse Assay, Western Blot

    KCa3.1 involved in microglia SOCE and ER stress. a , b Representative images of GRP78, p-PERK, and p-eIF2α in KCa3.1 −/− microglia, responses to 500 μM MPP + ( a ) or 1 μM Tg ( b ) vs. WT cells. Mean values of GRP78, p-PERK, and p-eIF2α relative to β-actin. Data are presented as the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results. * p
    Figure Legend Snippet: KCa3.1 involved in microglia SOCE and ER stress. a , b Representative images of GRP78, p-PERK, and p-eIF2α in KCa3.1 −/− microglia, responses to 500 μM MPP + ( a ) or 1 μM Tg ( b ) vs. WT cells. Mean values of GRP78, p-PERK, and p-eIF2α relative to β-actin. Data are presented as the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results. * p

    Techniques Used: Western Blot

    Upregulation of KCa3.1 channels and Iba1 in the brains of PD mouse model. a Western blot analysis of SNpc lysates from control and MPTP-induced PD mouse model analyzed by antibodies to TH, GFAP, Iba1, and KCa3.1. Data represent the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results; * p
    Figure Legend Snippet: Upregulation of KCa3.1 channels and Iba1 in the brains of PD mouse model. a Western blot analysis of SNpc lysates from control and MPTP-induced PD mouse model analyzed by antibodies to TH, GFAP, Iba1, and KCa3.1. Data represent the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results; * p

    Techniques Used: Western Blot

    Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced loss of DA neurons. a – g WT or KCa3.1 −/− mice received sequential intraperitoneal injections of MPTP (20 mg/kg) with or without senicapoc (100 mg/kg, once daily, p.o.) treatment for 5 days as described in the “ Material and methods ” section. Open field test ( b – e ) and the rotarod test ( f , g ) for bradykinesia were performed. Behavioral tests for MPTP-induced bradykinesia were conducted on the indicated days. Data are presented as mean ± SEM ( n = 10–15). b – e ** p
    Figure Legend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced loss of DA neurons. a – g WT or KCa3.1 −/− mice received sequential intraperitoneal injections of MPTP (20 mg/kg) with or without senicapoc (100 mg/kg, once daily, p.o.) treatment for 5 days as described in the “ Material and methods ” section. Open field test ( b – e ) and the rotarod test ( f , g ) for bradykinesia were performed. Behavioral tests for MPTP-induced bradykinesia were conducted on the indicated days. Data are presented as mean ± SEM ( n = 10–15). b – e ** p

    Techniques Used: Mouse Assay

    Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuated MPTP-induced ER stress. a , d Western blot analysis of GRP78 and CHOP protein levels in SNpc. b , c , e , f Data are presented as the mean ± SEM ( n = 5–6). Western blot was repeated three times and showed similar results. # p
    Figure Legend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuated MPTP-induced ER stress. a , d Western blot analysis of GRP78 and CHOP protein levels in SNpc. b , c , e , f Data are presented as the mean ± SEM ( n = 5–6). Western blot was repeated three times and showed similar results. # p

    Techniques Used: Western Blot

    Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced microgliosis. a , c Immunostaining for Iba1 in SNpc. Bar 50 μM. Quantitative analysis of Iba1 + cells in SNpc. Data are presented as mean ± SEM ( n = 5–8). * p
    Figure Legend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced microgliosis. a , c Immunostaining for Iba1 in SNpc. Bar 50 μM. Quantitative analysis of Iba1 + cells in SNpc. Data are presented as mean ± SEM ( n = 5–8). * p

    Techniques Used: Immunostaining

    3) Product Images from "Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling"

    Article Title: Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1351-x

    AKT modulation is crucial for KCa3.1-mediated ER stress in astrocytes. a Representative blots of p-AKT and total AKT from the hippocampi of postmortem human AD patients and age-matched controls. b Data are presented as the mean ± SEM ( n = 3–5). The OD value of p-AKT was normalized to that of AKT. ** p
    Figure Legend Snippet: AKT modulation is crucial for KCa3.1-mediated ER stress in astrocytes. a Representative blots of p-AKT and total AKT from the hippocampi of postmortem human AD patients and age-matched controls. b Data are presented as the mean ± SEM ( n = 3–5). The OD value of p-AKT was normalized to that of AKT. ** p

    Techniques Used:

    Elimination of KCa3.1 in APP/PS1 mice rescues spatial memory deficits in the MWM test. MWM testing of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice was performed as described in the “ Materials and methods ” section. a Escape latency. b Number of crossing the target quadrant by each group during the probe trials (no platform). c Percentage of swimming time spent in the target quadrant by each group during the probe trials (no platform). d Percentage of swimming distance spent in the target quadrant by each group during the probe trials (no platform). Data are presented as the mean ± SEM ( n = 10–12). # p
    Figure Legend Snippet: Elimination of KCa3.1 in APP/PS1 mice rescues spatial memory deficits in the MWM test. MWM testing of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice was performed as described in the “ Materials and methods ” section. a Escape latency. b Number of crossing the target quadrant by each group during the probe trials (no platform). c Percentage of swimming time spent in the target quadrant by each group during the probe trials (no platform). d Percentage of swimming distance spent in the target quadrant by each group during the probe trials (no platform). Data are presented as the mean ± SEM ( n = 10–12). # p

    Techniques Used: Mouse Assay

    Neuronal loss is rescued in brains of KCa3.1 −/− /APP/PS1 mice. a Immunofluorescence analysis of NeuN levels in the hippocampi of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b Quantification of neuron number/0.01 mm 2 in the hippocampus ( n = 6). Data are presented as the mean ± SEM. * p
    Figure Legend Snippet: Neuronal loss is rescued in brains of KCa3.1 −/− /APP/PS1 mice. a Immunofluorescence analysis of NeuN levels in the hippocampi of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b Quantification of neuron number/0.01 mm 2 in the hippocampus ( n = 6). Data are presented as the mean ± SEM. * p

    Techniques Used: Mouse Assay, Immunofluorescence

    Decreased neuroinflammation in brains of KCa3.1 −/− /APP/PS1 mice. a Levels of activated microglia in CA1 areas of the mouse hippocampus were analyzed by immunostaining of the microglia marker Iba1. b Quantification of activated microglia number/0.01 mm 2 in the hippocampus ( n = 3). Data are presented as the mean ± SEM. * p
    Figure Legend Snippet: Decreased neuroinflammation in brains of KCa3.1 −/− /APP/PS1 mice. a Levels of activated microglia in CA1 areas of the mouse hippocampus were analyzed by immunostaining of the microglia marker Iba1. b Quantification of activated microglia number/0.01 mm 2 in the hippocampus ( n = 3). Data are presented as the mean ± SEM. * p

    Techniques Used: Mouse Assay, Immunostaining, Marker

    KCa3.1 upregulation in Aβ-induced RA and the brains of AD patients. a Primary astrocytes were stimulated with 5 μM Aβ and lysates were subjected to Western blot analysis with antibodies against KCa3.1, Orai1, and STIM1. β-actin was used to confirm equal loading. b Data are presented as the mean ± SEM ( n = 5). The OD values of KCa3.1, Orai1, and STIM1 were normalized to that of β-actin. * p
    Figure Legend Snippet: KCa3.1 upregulation in Aβ-induced RA and the brains of AD patients. a Primary astrocytes were stimulated with 5 μM Aβ and lysates were subjected to Western blot analysis with antibodies against KCa3.1, Orai1, and STIM1. β-actin was used to confirm equal loading. b Data are presented as the mean ± SEM ( n = 5). The OD values of KCa3.1, Orai1, and STIM1 were normalized to that of β-actin. * p

    Techniques Used: Western Blot

    KCa3.1 contributes to increased ER stress in APP/PS1 mice. a Western blot analysis of GRP78 and CHOP protein levels in hippocampal extracts of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b , c Data are presented as the mean ± SEM ( n = 3–5). The OD values of GRP78 ( b ) and CHOP ( c ) were normalized to that of β-actin. # p
    Figure Legend Snippet: KCa3.1 contributes to increased ER stress in APP/PS1 mice. a Western blot analysis of GRP78 and CHOP protein levels in hippocampal extracts of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b , c Data are presented as the mean ± SEM ( n = 3–5). The OD values of GRP78 ( b ) and CHOP ( c ) were normalized to that of β-actin. # p

    Techniques Used: Mouse Assay, Western Blot

    KCa3.1 involved in astrocytes SOCE and ER stress. a Primary cultured astrocytes were treated with 5 μM Aβ for 12 h with or without pretreatment of the KCa3.1 blocker TRAM-34 (1 μM). Fluorescence intensities of [Ca 2+ ] i are shown. Fluorescence intensity was measured in the presence of 1 μM Tg with or without 2 mM Ca 2+ . b Data are presented as the mean ± SEM ( n = 10). # p
    Figure Legend Snippet: KCa3.1 involved in astrocytes SOCE and ER stress. a Primary cultured astrocytes were treated with 5 μM Aβ for 12 h with or without pretreatment of the KCa3.1 blocker TRAM-34 (1 μM). Fluorescence intensities of [Ca 2+ ] i are shown. Fluorescence intensity was measured in the presence of 1 μM Tg with or without 2 mM Ca 2+ . b Data are presented as the mean ± SEM ( n = 10). # p

    Techniques Used: Cell Culture, Fluorescence

    4) Product Images from "Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells"

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154471

    Functional expression of SK4 channels in breast cancer cells. (A) Immunoblotting of SK4 and EMT-related proteins (E-cadherin and Vimentin) in breast cancer cell lines. (B) Comparison of SK4 mRNA expression in 4 breast cancer cell lines as determined by real-time PCR; n = 3. (C) Immunoblotting of ER protein in MDA-MB-468, MDA-MB-231 and T47D cells. (D-G) Immunostaining of SK4 (red) and nuclear marker DAPI (blue) in MDA-MB-231 (D), MDA-MB-468 (E), MCF-7 (F) and T47D (G) cells. Scale bars, 50 μm. (H, I) Whole-cell recording of MDA-MB-231 cells before (H) and after (I) 5-μM TRAM-34 treatment. (J, K) With (J) or without (K) 350 nM free Ca 2+ in the pipette solution, the voltage-current density curves show the currents changes before (a) and after (b) TRAM-34 treatment. The currents were evoked by step voltage ranging from -100 mV to +100 mV in steps of 10 mV every 100 ms. Dunnett’s Multiple Comparison Test was applied in comparison, ** p
    Figure Legend Snippet: Functional expression of SK4 channels in breast cancer cells. (A) Immunoblotting of SK4 and EMT-related proteins (E-cadherin and Vimentin) in breast cancer cell lines. (B) Comparison of SK4 mRNA expression in 4 breast cancer cell lines as determined by real-time PCR; n = 3. (C) Immunoblotting of ER protein in MDA-MB-468, MDA-MB-231 and T47D cells. (D-G) Immunostaining of SK4 (red) and nuclear marker DAPI (blue) in MDA-MB-231 (D), MDA-MB-468 (E), MCF-7 (F) and T47D (G) cells. Scale bars, 50 μm. (H, I) Whole-cell recording of MDA-MB-231 cells before (H) and after (I) 5-μM TRAM-34 treatment. (J, K) With (J) or without (K) 350 nM free Ca 2+ in the pipette solution, the voltage-current density curves show the currents changes before (a) and after (b) TRAM-34 treatment. The currents were evoked by step voltage ranging from -100 mV to +100 mV in steps of 10 mV every 100 ms. Dunnett’s Multiple Comparison Test was applied in comparison, ** p

    Techniques Used: Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Immunostaining, Marker, Transferring, Mass Spectrometry

    Blockage of SK4 channels inhibits MDA-MB-231 cell proliferation and colony formation ability, but not that of T47D cells. (A-D) Cell growth of MDA-MB-231 (A, B) and T47D cells (C, D) was analyzed using an MTT assay. The two cell lines were treated with 0–20 μM TRAM-34 (A, C) or clotrimazole (B, D) for 48 h, and the absorbance was measured; n = 5. (E, F) Images of the formed MDA-MB-231 colonies in the control group (CTL) and treatment groups (10 μM TRAM-34 and 20 μM TRAM-34); the bar represents separate counts of the colonies; n = 4. The data are presented as the mean ± SD, and Dunnett’s Multiple Comparison Test was applied in comparison. * p
    Figure Legend Snippet: Blockage of SK4 channels inhibits MDA-MB-231 cell proliferation and colony formation ability, but not that of T47D cells. (A-D) Cell growth of MDA-MB-231 (A, B) and T47D cells (C, D) was analyzed using an MTT assay. The two cell lines were treated with 0–20 μM TRAM-34 (A, C) or clotrimazole (B, D) for 48 h, and the absorbance was measured; n = 5. (E, F) Images of the formed MDA-MB-231 colonies in the control group (CTL) and treatment groups (10 μM TRAM-34 and 20 μM TRAM-34); the bar represents separate counts of the colonies; n = 4. The data are presented as the mean ± SD, and Dunnett’s Multiple Comparison Test was applied in comparison. * p

    Techniques Used: Multiple Displacement Amplification, MTT Assay, CTL Assay

    Blockage of SK4 channels promotes apoptosis in MDA-MB-231 cells but not T47D cells. MDA-MB-231 (A, B) and T47D (C, D) cells were treated with 20 μM TRAM-34 for 24 or 48 h, and cell apoptosis was analyzed by Annexin V-FITC/ PI-PE staining and flow cytometry. The bar of MDA-MB-231 indicates that the apoptosis rate of the TRAM-34-treated group increased apparently compared with that of the control (CTL). For T47D, the difference was not significant. The data are presented as the mean ± SD, and unpaired t test was applied in comparison. n = 3; * p
    Figure Legend Snippet: Blockage of SK4 channels promotes apoptosis in MDA-MB-231 cells but not T47D cells. MDA-MB-231 (A, B) and T47D (C, D) cells were treated with 20 μM TRAM-34 for 24 or 48 h, and cell apoptosis was analyzed by Annexin V-FITC/ PI-PE staining and flow cytometry. The bar of MDA-MB-231 indicates that the apoptosis rate of the TRAM-34-treated group increased apparently compared with that of the control (CTL). For T47D, the difference was not significant. The data are presented as the mean ± SD, and unpaired t test was applied in comparison. n = 3; * p

    Techniques Used: Multiple Displacement Amplification, Staining, Flow Cytometry, Cytometry, CTL Assay

    Down-regulation of SK4 channels inhibits the migration of MDA-MB-231 cells. A negative control siRNA (N.C.) and 3 SK4-specific siRNAs (Si-1, Si-2 and Si-3) were transfected into MDA-MB-231 cells, and 20 μM TRAM-34 was added to the TRAM-34-treated group to inhibit SK4 channels. (A, B) Knockdown of SK4 by siRNA was confirmed using immunoblotting and real-time PCR; n = 3. (C, D) The images and bar of the transwell migration assay indicate that the counts of migrated cells in SK4 siRNA (Si-SK4)- and TRAM-34-treated group were significantly less than those of the control (CTL). Scale bars, 50 μm; n = 4. (E, F) The images and bar of the wound-healing assay. The wound-healing rate represents the distance migrated by cells at certain time divided by the wound distance at 0 h. Scale bars, 100 μm; n = 3. The data are presented as the mean ± SD, Dunnett’s Multiple Comparison Test was applied in (B) and (D), and unpaired t test in (F). ** p
    Figure Legend Snippet: Down-regulation of SK4 channels inhibits the migration of MDA-MB-231 cells. A negative control siRNA (N.C.) and 3 SK4-specific siRNAs (Si-1, Si-2 and Si-3) were transfected into MDA-MB-231 cells, and 20 μM TRAM-34 was added to the TRAM-34-treated group to inhibit SK4 channels. (A, B) Knockdown of SK4 by siRNA was confirmed using immunoblotting and real-time PCR; n = 3. (C, D) The images and bar of the transwell migration assay indicate that the counts of migrated cells in SK4 siRNA (Si-SK4)- and TRAM-34-treated group were significantly less than those of the control (CTL). Scale bars, 50 μm; n = 4. (E, F) The images and bar of the wound-healing assay. The wound-healing rate represents the distance migrated by cells at certain time divided by the wound distance at 0 h. Scale bars, 100 μm; n = 3. The data are presented as the mean ± SD, Dunnett’s Multiple Comparison Test was applied in (B) and (D), and unpaired t test in (F). ** p

    Techniques Used: Migration, Multiple Displacement Amplification, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Transwell Migration Assay, CTL Assay, Wound Healing Assay

    The EGF/bFGF-induced EMT of MDA-MB-231 cells correlates with SK4 channels. (A) Phase contrast images of MDA-231 and T47D cells treated with (E+b) or without (CTL) EGF/bFGF for 1 day, 3 days and 5 days. Scale bars, 100 μm. (B, C) The EGF/bFGF-induced EMT of MDA-231 cells was confirmed using immunoblotting and real-time PCR of EMT markers (Vimentin, Snail1 and Slug), and the SK4 mRNA level increased after EMT. (D) Immunoblotting of EMT-related proteins (Vimentin and Snail1) was performed 72 h after MDA-231 cells were transfected with negative control siRNA (N.C.) or SK4-specific siRNA (Si-SK4); cells that did not undergo transfection served as a control (CTL). The data are presented as the mean ± SD, and paired t test was applied in comparison. n = 3; * p
    Figure Legend Snippet: The EGF/bFGF-induced EMT of MDA-MB-231 cells correlates with SK4 channels. (A) Phase contrast images of MDA-231 and T47D cells treated with (E+b) or without (CTL) EGF/bFGF for 1 day, 3 days and 5 days. Scale bars, 100 μm. (B, C) The EGF/bFGF-induced EMT of MDA-231 cells was confirmed using immunoblotting and real-time PCR of EMT markers (Vimentin, Snail1 and Slug), and the SK4 mRNA level increased after EMT. (D) Immunoblotting of EMT-related proteins (Vimentin and Snail1) was performed 72 h after MDA-231 cells were transfected with negative control siRNA (N.C.) or SK4-specific siRNA (Si-SK4); cells that did not undergo transfection served as a control (CTL). The data are presented as the mean ± SD, and paired t test was applied in comparison. n = 3; * p

    Techniques Used: Multiple Displacement Amplification, CTL Assay, Real-time Polymerase Chain Reaction, Transfection, Negative Control

    SK4 proteins expressed in breast cancer tissue. (A-D) SK4 IHC in fours subtypes of breast cancer tissues including Luminal A (A), Luminal B (B), HER2 (C), and TNBC (D). Scale bars, 50 μm. (E) Immunoblotting of SK4 and E-cadherin in breast cancer tissues (BC1 and BC2) and non-tumor breast tissues (Non-Tumor1 and Non-Tumor2).
    Figure Legend Snippet: SK4 proteins expressed in breast cancer tissue. (A-D) SK4 IHC in fours subtypes of breast cancer tissues including Luminal A (A), Luminal B (B), HER2 (C), and TNBC (D). Scale bars, 50 μm. (E) Immunoblotting of SK4 and E-cadherin in breast cancer tissues (BC1 and BC2) and non-tumor breast tissues (Non-Tumor1 and Non-Tumor2).

    Techniques Used: Immunohistochemistry

    5) Product Images from "Functional Cooperation between KCa3.1 and TRPV4 Channels in Bronchial Smooth Muscle Cell Proliferation Associated with Chronic Asthma"

    Article Title: Functional Cooperation between KCa3.1 and TRPV4 Channels in Bronchial Smooth Muscle Cell Proliferation Associated with Chronic Asthma

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00559

    Gene deletion of KCa3.1 prevented established airway remodeling. (A) Representative images were obtained of Masson trichrome-stained lung sections from WT, KO, WT + OVA, and KO + OVA groups (original magnification, X 30). (B) Random measurements from the basement membrane into the submucosa (10 measurements of 20 μm in length) were taken, and the mean density was calculated from four bronchi per mouse ( n = 5). ∗∗ p
    Figure Legend Snippet: Gene deletion of KCa3.1 prevented established airway remodeling. (A) Representative images were obtained of Masson trichrome-stained lung sections from WT, KO, WT + OVA, and KO + OVA groups (original magnification, X 30). (B) Random measurements from the basement membrane into the submucosa (10 measurements of 20 μm in length) were taken, and the mean density was calculated from four bronchi per mouse ( n = 5). ∗∗ p

    Techniques Used: Staining

    KCa3.1 and TRPV4 involvement in HBSM cells proliferation. (A) Representative western blot showing the effect of siRNAs directed against KCa3.1 and TRPV4 on the protein level of KCa3.1. (B) Representative western blot showing the effect of siRNAs directed against KCa3.1 and TRPV4 on the protein level of TRPV4. (C) Analysis of HBSM cells proliferation transfected with siCTL, siKCa3.1, or siTRPV4. Cell proliferation (CCK-8 assay) was measured as described in methods 72 h post-transfection. Values were reported as means ± SEM normalized to the control ( n = 4). ∗∗ p
    Figure Legend Snippet: KCa3.1 and TRPV4 involvement in HBSM cells proliferation. (A) Representative western blot showing the effect of siRNAs directed against KCa3.1 and TRPV4 on the protein level of KCa3.1. (B) Representative western blot showing the effect of siRNAs directed against KCa3.1 and TRPV4 on the protein level of TRPV4. (C) Analysis of HBSM cells proliferation transfected with siCTL, siKCa3.1, or siTRPV4. Cell proliferation (CCK-8 assay) was measured as described in methods 72 h post-transfection. Values were reported as means ± SEM normalized to the control ( n = 4). ∗∗ p

    Techniques Used: Western Blot, Transfection, CCK-8 Assay

    KCa3.1 and TRPV4 involvement in HBSM cells proliferation via JNK/c-Jun pathways. (A) p-JNK, p-c-Jun, and REST protein expressions in 1h PDGF stimulated HBSM cells with or without pretreatment of TRAM-34 (1 μM) or HC 067047 (10 μM). (B) Data were presented as means ± SEM. n = 5, ∗ p
    Figure Legend Snippet: KCa3.1 and TRPV4 involvement in HBSM cells proliferation via JNK/c-Jun pathways. (A) p-JNK, p-c-Jun, and REST protein expressions in 1h PDGF stimulated HBSM cells with or without pretreatment of TRAM-34 (1 μM) or HC 067047 (10 μM). (B) Data were presented as means ± SEM. n = 5, ∗ p

    Techniques Used:

    Gene deletion of KCa3.1 reduced established airway inflammation. (A) Representative images of hematoxylin and eosin-stained lung sections from WT, KO, WT + OVA, and KO + OVA groups (original magnification, X30). (B) Quantitative analyses of inflammatory cell infiltration in lung sections. Data were expressed as means ± SEM ( n = 5). ∗∗ p
    Figure Legend Snippet: Gene deletion of KCa3.1 reduced established airway inflammation. (A) Representative images of hematoxylin and eosin-stained lung sections from WT, KO, WT + OVA, and KO + OVA groups (original magnification, X30). (B) Quantitative analyses of inflammatory cell infiltration in lung sections. Data were expressed as means ± SEM ( n = 5). ∗∗ p

    Techniques Used: Staining

    Gene deletion of KCa3.1 prevented OVA-induced up-regulation of α-SMA. (A) The KCa3.1 and TRPV4 proteins expressions in the lung sections from WT and WT + OVA groups mice. (B) Data were presented as means ± SEM ( n = 5). ∗ p
    Figure Legend Snippet: Gene deletion of KCa3.1 prevented OVA-induced up-regulation of α-SMA. (A) The KCa3.1 and TRPV4 proteins expressions in the lung sections from WT and WT + OVA groups mice. (B) Data were presented as means ± SEM ( n = 5). ∗ p

    Techniques Used: Mouse Assay

    TRPV4 involved in Ca 2+ entry induced by KCa3.1 activation in asthmatic HBSM cells. (A,C) Representative curves showed the fold changes in [Ca 2+ ] i relative to the unstimulated condition over 360 s. (B,D) The bar graphs indicated the maximal increase in [Ca 2+ ] i mobilization. (A) External calcium was required for 200 μM 1-EBIO-induced Ca 2+ elevations. The increase of [Ca 2+ ] i induced by 200 μM 1-EBIO was prevented by 0 external Ca 2+ . (B) Quantitative analysis of 1-EBIO-induced Ca 2+ entry. n = 10–20, ∗ p
    Figure Legend Snippet: TRPV4 involved in Ca 2+ entry induced by KCa3.1 activation in asthmatic HBSM cells. (A,C) Representative curves showed the fold changes in [Ca 2+ ] i relative to the unstimulated condition over 360 s. (B,D) The bar graphs indicated the maximal increase in [Ca 2+ ] i mobilization. (A) External calcium was required for 200 μM 1-EBIO-induced Ca 2+ elevations. The increase of [Ca 2+ ] i induced by 200 μM 1-EBIO was prevented by 0 external Ca 2+ . (B) Quantitative analysis of 1-EBIO-induced Ca 2+ entry. n = 10–20, ∗ p

    Techniques Used: Activation Assay

    Gene deletion of KCa3.1 reduced established OVA-induced changes in constrictor responsiveness to methacholine in mouse bronchi. Relative to WT group bronchi, maximal isometric contractile force responses to methacholine were significantly increased in WT + OVA group bronchi, and this increased constrictor responsiveness to methacholine was prevented in KO + OVA group bronchi tissues. Data represented means ± SEM ( n = 4–6). # p
    Figure Legend Snippet: Gene deletion of KCa3.1 reduced established OVA-induced changes in constrictor responsiveness to methacholine in mouse bronchi. Relative to WT group bronchi, maximal isometric contractile force responses to methacholine were significantly increased in WT + OVA group bronchi, and this increased constrictor responsiveness to methacholine was prevented in KO + OVA group bronchi tissues. Data represented means ± SEM ( n = 4–6). # p

    Techniques Used:

    KCa3.1 and TRPV4 appear colocalized in asthmatic HBSM cells. (A,B) Double immunofluorescence images of KCa3.1 (green) and TRPV4 (red) in HBSM cells. Note the strong colocalization indicated by merge yellow fluorescence, quantification of the colocalization observed in experiments as shown in (C) . (C) The histograms represent the ratio of the mean Pearson correlation coefficient calculated from the colabeling in a number of samples, as indicated above the bar. Scale bar: 25 μm.
    Figure Legend Snippet: KCa3.1 and TRPV4 appear colocalized in asthmatic HBSM cells. (A,B) Double immunofluorescence images of KCa3.1 (green) and TRPV4 (red) in HBSM cells. Note the strong colocalization indicated by merge yellow fluorescence, quantification of the colocalization observed in experiments as shown in (C) . (C) The histograms represent the ratio of the mean Pearson correlation coefficient calculated from the colabeling in a number of samples, as indicated above the bar. Scale bar: 25 μm.

    Techniques Used: Immunofluorescence, Fluorescence

    6) Product Images from "Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury"

    Article Title: Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury

    Journal: Acta Pharmaceutica Sinica. B

    doi: 10.1016/j.apsb.2020.05.002

    Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P
    Figure Legend Snippet: Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P

    Techniques Used: Mouse Assay, Expressing, Immunostaining, Staining, Marker, MANN-WHITNEY

    A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.
    Figure Legend Snippet: A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.

    Techniques Used: Binding Assay

    7) Product Images from "Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1"

    Article Title: Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1

    Journal: eLife

    doi: 10.7554/eLife.16093

    TTM activates and copper inhibits KCa3.1 in inside-out membrane patches, dependent on His358 phosphorylation. Representative recordings of channel activity versus time from I/O patches isolated from 293-KCa3.1 ( A – F ) or parental 293 ( G ) cells. All recordings were done in 300 nM free Ca 2+ and 500 µM GTP unless otherwise specified. TTM (20 µM), CuCl 2 (10 µM), NDPK-B (10 µg/ml) and TRAM-34 (1 µM) were added as indicated. Shown are representative patch data from three independent experiments. ( A – C ) were performed in the presence of EGTA (5 mM) and ( D – F ) in the absence of EGTA in the bath solution. ( E , F ) were done in the presence of 10 µM free calcium in the bath solution. DOI: http://dx.doi.org/10.7554/eLife.16093.003
    Figure Legend Snippet: TTM activates and copper inhibits KCa3.1 in inside-out membrane patches, dependent on His358 phosphorylation. Representative recordings of channel activity versus time from I/O patches isolated from 293-KCa3.1 ( A – F ) or parental 293 ( G ) cells. All recordings were done in 300 nM free Ca 2+ and 500 µM GTP unless otherwise specified. TTM (20 µM), CuCl 2 (10 µM), NDPK-B (10 µg/ml) and TRAM-34 (1 µM) were added as indicated. Shown are representative patch data from three independent experiments. ( A – C ) were performed in the presence of EGTA (5 mM) and ( D – F ) in the absence of EGTA in the bath solution. ( E , F ) were done in the presence of 10 µM free calcium in the bath solution. DOI: http://dx.doi.org/10.7554/eLife.16093.003

    Techniques Used: Activity Assay, Isolation

    CD4 + T cells from Ctr1 +/ - mice are hyperactivated. Naive CD4 + T cells were isolated from spleens of wild-type (WT) or Ctr1 +/- mice and activated with anti-CD3 and -CD28 antibodies for 48 hr. ( A ) KCa3.1 channel activity was determined by whole-cell patch clamping as in Figure 1 . Shown is a summary bar graph of TRAM-34 (1 µM; KCa3.1) and ShK (1 nM; Kv1.3)-sensitive currents at +60 mV (n = 12 (WT) or 15 ( Ctr1 +/- )). For statistical analysis, one-way-ANOVA was used, and the Bonferroni test was applied to compare the mean values. Data are displayed as mean ± SEM (n = 10 cells). *p≤0.01 vs TRAM-34-sensitive current in WT. ( B ) KCa3.1 was immunoprecipitated from lysates of CD4 + T cells from WT or Ctr1 +/- mice and probed with a monoclonal antibody to 3-pHis (clone SC56-2) or KCa3.1 as indicated. ( C ) Activated cells were rested overnight, loaded with Fura-2, AM and attached to a poly-L-lysine-coated coverslip for 20 min. Calcium imaging was then performed in unstimulated cells and following stimulation with anti-CD3 and -CD28 antibodies (n = 80–100 cells). ( D ) ELISA to quantify IL-2 and IFN-γ in the supernatants. Statistical significance was calculated using Student’s t -test (*p
    Figure Legend Snippet: CD4 + T cells from Ctr1 +/ - mice are hyperactivated. Naive CD4 + T cells were isolated from spleens of wild-type (WT) or Ctr1 +/- mice and activated with anti-CD3 and -CD28 antibodies for 48 hr. ( A ) KCa3.1 channel activity was determined by whole-cell patch clamping as in Figure 1 . Shown is a summary bar graph of TRAM-34 (1 µM; KCa3.1) and ShK (1 nM; Kv1.3)-sensitive currents at +60 mV (n = 12 (WT) or 15 ( Ctr1 +/- )). For statistical analysis, one-way-ANOVA was used, and the Bonferroni test was applied to compare the mean values. Data are displayed as mean ± SEM (n = 10 cells). *p≤0.01 vs TRAM-34-sensitive current in WT. ( B ) KCa3.1 was immunoprecipitated from lysates of CD4 + T cells from WT or Ctr1 +/- mice and probed with a monoclonal antibody to 3-pHis (clone SC56-2) or KCa3.1 as indicated. ( C ) Activated cells were rested overnight, loaded with Fura-2, AM and attached to a poly-L-lysine-coated coverslip for 20 min. Calcium imaging was then performed in unstimulated cells and following stimulation with anti-CD3 and -CD28 antibodies (n = 80–100 cells). ( D ) ELISA to quantify IL-2 and IFN-γ in the supernatants. Statistical significance was calculated using Student’s t -test (*p

    Techniques Used: Mouse Assay, Isolation, Activity Assay, Immunoprecipitation, Imaging, Enzyme-linked Immunosorbent Assay

    Calcium is required for activation of KCa3.1 by NDPK-B or TTM in inside-out membrane patches. Representative recordings of channel activity versus time from I/O patches isolated from 293-KCa3.1 cells as described in Figure 2 . All recordings were done in 500 µM GTP and either 30 or 300 nM free Ca 2+ as indicated. TTM (20 µM), NDPK-B (10 µg/ml), CuCl 2 (10 µM) and TRAM-34 (1 µM) were added as indicated. Shown are representative patch data from three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.16093.004
    Figure Legend Snippet: Calcium is required for activation of KCa3.1 by NDPK-B or TTM in inside-out membrane patches. Representative recordings of channel activity versus time from I/O patches isolated from 293-KCa3.1 cells as described in Figure 2 . All recordings were done in 500 µM GTP and either 30 or 300 nM free Ca 2+ as indicated. TTM (20 µM), NDPK-B (10 µg/ml), CuCl 2 (10 µM) and TRAM-34 (1 µM) were added as indicated. Shown are representative patch data from three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.16093.004

    Techniques Used: Activation Assay, Activity Assay, Isolation

    Basal KCa3.1 current is elevated in transfected MEFs from Ctr1 -/- mice. ( A , B ) Representative current vs. voltage (IV) plot recorded from a KCa3.1-transfected MEF from a wild-type (WT) mouse ( A ) or a Ctr1 -/- ( B ) mouse. After obtaining the basal current, TTM (20 µM) was perfused in the bath solution followed by CuCl 2 (10 µM) and then TRAM-34 (1 µM). ( C ) Summary bar graph of the TRAM-34-sensitive current at +60 mV for data measured from MEFs from WT or Ctr1 -/- mice. For statistical analysis, one-way-ANOVA was used, and the Bonferroni test was applied to compare the mean values. Data are displayed as mean ± SEM (n = 10 cells). *p≤0.01 versus Basal in WT MEFs and for +CuCl 2 versus +TTM. ( D ) Exofacial HA-tagged KCa3.1 was transfected into WT or Ctr1 -/- MEFs, and cell surface expression was assessed by FACs analysis following staining with anti-HA antibodies and with anti-mouse FITC antibodies in non-permeabilized cells. (i) Flow cytometry results of WT and Ctr1 -/- controls stained with only the secondary anti-mouse FITC antibody, or of WT + KCa3.1-HA and Ctr1 -/- + KCa3.1-HA MEFs stained with both anti-HA and anti-mouse FITC antibodies. (ii) Mean fluorescence intensity (MFI) of WT + KCa3.1-HA and Ctr1 -/- + KCa3.1-HA MEFs. DOI: http://dx.doi.org/10.7554/eLife.16093.005
    Figure Legend Snippet: Basal KCa3.1 current is elevated in transfected MEFs from Ctr1 -/- mice. ( A , B ) Representative current vs. voltage (IV) plot recorded from a KCa3.1-transfected MEF from a wild-type (WT) mouse ( A ) or a Ctr1 -/- ( B ) mouse. After obtaining the basal current, TTM (20 µM) was perfused in the bath solution followed by CuCl 2 (10 µM) and then TRAM-34 (1 µM). ( C ) Summary bar graph of the TRAM-34-sensitive current at +60 mV for data measured from MEFs from WT or Ctr1 -/- mice. For statistical analysis, one-way-ANOVA was used, and the Bonferroni test was applied to compare the mean values. Data are displayed as mean ± SEM (n = 10 cells). *p≤0.01 versus Basal in WT MEFs and for +CuCl 2 versus +TTM. ( D ) Exofacial HA-tagged KCa3.1 was transfected into WT or Ctr1 -/- MEFs, and cell surface expression was assessed by FACs analysis following staining with anti-HA antibodies and with anti-mouse FITC antibodies in non-permeabilized cells. (i) Flow cytometry results of WT and Ctr1 -/- controls stained with only the secondary anti-mouse FITC antibody, or of WT + KCa3.1-HA and Ctr1 -/- + KCa3.1-HA MEFs stained with both anti-HA and anti-mouse FITC antibodies. (ii) Mean fluorescence intensity (MFI) of WT + KCa3.1-HA and Ctr1 -/- + KCa3.1-HA MEFs. DOI: http://dx.doi.org/10.7554/eLife.16093.005

    Techniques Used: Transfection, Mouse Assay, Expressing, FACS, Staining, Flow Cytometry, Cytometry, Fluorescence

    Metal chelators activate and copper inhibits KCa3.1 in whole-cell membrane patches. ( A – D ) (i) Representative current vs. voltage (IV) plot recorded from a 293-KCa3.1(WT) cell ( A – C ) or a 293-KCa3.1(H358N) cell (D). After obtaining the basal current, TPEN (20 µM) ( A , B ) or TTM (20 µM) ( C , D ) was perfused in the bath solution, followed by the addition of either ZnCl 2 (100 µM) ( A ) or CuCl 2 (100 µM) ( B – D ), and then TRAM-34 (1 µM). ( A – D ) (ii) Representative trace of current (pA/pF) recorded at +60 mV using ramp protocol applied every 10 s (sweep frequency). The timing of additions (TPEN, CuCl 2 , etc.) is indicated by the arrows. ( E , F ) Summary bar graph of TRAM-34-sensitive current at +60 mV for data measured from 293-KCa3.1(WT) cells ( E ) or from 293-KCa3.1(H358N) cells ( F ). In ( E ), +ZnCl 2 is to be compared with +TPEN (i.e. ZnCl 2 added after treatment with TPEN), and +CuCl 2 with +TTM (i.e. CuCl 2 added after treatment with TTM). Data are displayed as mean ± the standard error of the mean (SEM) (n = 10 ( E ) or 8 ( F ) cells). *p≤0.01 versus Basal and for +CuCl 2 versus +TTM. DOI: http://dx.doi.org/10.7554/eLife.16093.002
    Figure Legend Snippet: Metal chelators activate and copper inhibits KCa3.1 in whole-cell membrane patches. ( A – D ) (i) Representative current vs. voltage (IV) plot recorded from a 293-KCa3.1(WT) cell ( A – C ) or a 293-KCa3.1(H358N) cell (D). After obtaining the basal current, TPEN (20 µM) ( A , B ) or TTM (20 µM) ( C , D ) was perfused in the bath solution, followed by the addition of either ZnCl 2 (100 µM) ( A ) or CuCl 2 (100 µM) ( B – D ), and then TRAM-34 (1 µM). ( A – D ) (ii) Representative trace of current (pA/pF) recorded at +60 mV using ramp protocol applied every 10 s (sweep frequency). The timing of additions (TPEN, CuCl 2 , etc.) is indicated by the arrows. ( E , F ) Summary bar graph of TRAM-34-sensitive current at +60 mV for data measured from 293-KCa3.1(WT) cells ( E ) or from 293-KCa3.1(H358N) cells ( F ). In ( E ), +ZnCl 2 is to be compared with +TPEN (i.e. ZnCl 2 added after treatment with TPEN), and +CuCl 2 with +TTM (i.e. CuCl 2 added after treatment with TTM). Data are displayed as mean ± the standard error of the mean (SEM) (n = 10 ( E ) or 8 ( F ) cells). *p≤0.01 versus Basal and for +CuCl 2 versus +TTM. DOI: http://dx.doi.org/10.7554/eLife.16093.002

    Techniques Used:

    Model for KCa3.1 activation by calcium and histidine phosphorylation. For clarity, only two of the four KCa3.1 subunits and calmodulin (CaM) are shown. In the basal state (no calcium; left panel), the CaM C lobe (green sphere) is bound to the N-terminal segment of the calmodulin-binding domain (CBD, yellow) in KCa3.1 ( Schumacher et al., 2004 ). The transmembrane S6 helices close off the channel on the cytoplasmic side. At the C terminus of KCa3.1 is a coiled-coil region that forms a four-helix bundle (violet cylinders; S.R.H., unpublished data). Based on coiled-coil prediction software, it is probable that the region containing His358, which is just C-terminal to the CBD, also forms a four-helix bundle (maroon cylinders), with His358 (pentagon) occupying an inward-facing ‘a’ position in the heptad repeat. This would position the four copies of His358 for coordination of a Cu(II) ion on the axis of the four-helix bundle, which would stabilize the four-helix bundle and act to resist the conformational changes induced by calcium binding to the CaM N lobe (blue sphere). An increase in intracellular calcium induces conformational changes in the CBD that partially destabilize copper binding (middle panel), providing access to His358 for phosphorylation by NDPK-B (or copper chelation by TTM). Upon phosphorylation of His358, copper binding is abrogated, and the calcium-induced conformational changes in the CBD lead to channel opening (right panel; exact mechanism not known) ( Adelman et al., 2012 ; Sachyani et al., 2014 ). DOI: http://dx.doi.org/10.7554/eLife.16093.007
    Figure Legend Snippet: Model for KCa3.1 activation by calcium and histidine phosphorylation. For clarity, only two of the four KCa3.1 subunits and calmodulin (CaM) are shown. In the basal state (no calcium; left panel), the CaM C lobe (green sphere) is bound to the N-terminal segment of the calmodulin-binding domain (CBD, yellow) in KCa3.1 ( Schumacher et al., 2004 ). The transmembrane S6 helices close off the channel on the cytoplasmic side. At the C terminus of KCa3.1 is a coiled-coil region that forms a four-helix bundle (violet cylinders; S.R.H., unpublished data). Based on coiled-coil prediction software, it is probable that the region containing His358, which is just C-terminal to the CBD, also forms a four-helix bundle (maroon cylinders), with His358 (pentagon) occupying an inward-facing ‘a’ position in the heptad repeat. This would position the four copies of His358 for coordination of a Cu(II) ion on the axis of the four-helix bundle, which would stabilize the four-helix bundle and act to resist the conformational changes induced by calcium binding to the CaM N lobe (blue sphere). An increase in intracellular calcium induces conformational changes in the CBD that partially destabilize copper binding (middle panel), providing access to His358 for phosphorylation by NDPK-B (or copper chelation by TTM). Upon phosphorylation of His358, copper binding is abrogated, and the calcium-induced conformational changes in the CBD lead to channel opening (right panel; exact mechanism not known) ( Adelman et al., 2012 ; Sachyani et al., 2014 ). DOI: http://dx.doi.org/10.7554/eLife.16093.007

    Techniques Used: Activation Assay, Chick Chorioallantoic Membrane Assay, Binding Assay, Software, Activated Clotting Time Assay

    8) Product Images from "Inhibition of the Ca2+-Dependent K+ Channel, KCNN4/KCa3.1, Improves Tissue Protection and Locomotor Recovery after Spinal Cord Injury"

    Article Title: Inhibition of the Ca2+-Dependent K+ Channel, KCNN4/KCa3.1, Improves Tissue Protection and Locomotor Recovery after Spinal Cord Injury

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0047-11.2011

    KCa3.1 immunoreactivity was not detected in macrophages and microglia in the injured mouse spinal cord. A–I , Double labeling with anti-KCa3.1 antibody ( A , D , G ) and different macrophage/microglial markers ( B , E , H ) in perfusion fixed tissue sections of the spinal cord at 7 d after SCI. The anti-Mac-2 antibody ( B ) recognizes phagocytic macrophages; tomato lectin ( E ) binds to macrophages and microglial cells; and Iba-1 ( H ) is upregulated in activated macrophages/microglia. The lesion core is to the right of the dashed lines in the merged images in C and F . Note that the macrophages/microglia in the lesion lack KCa3.1 staining. J–L , Unfixed tissue section of the spinal cord taken at 7 dpi was double labeled for KCa3.1 ( J ) and CD11b (Mac-1 antibody; K ). Note the absence of double labeling of the Mac-1 + macrophages ( L ). Scale bars, 50 μm.
    Figure Legend Snippet: KCa3.1 immunoreactivity was not detected in macrophages and microglia in the injured mouse spinal cord. A–I , Double labeling with anti-KCa3.1 antibody ( A , D , G ) and different macrophage/microglial markers ( B , E , H ) in perfusion fixed tissue sections of the spinal cord at 7 d after SCI. The anti-Mac-2 antibody ( B ) recognizes phagocytic macrophages; tomato lectin ( E ) binds to macrophages and microglial cells; and Iba-1 ( H ) is upregulated in activated macrophages/microglia. The lesion core is to the right of the dashed lines in the merged images in C and F . Note that the macrophages/microglia in the lesion lack KCa3.1 staining. J–L , Unfixed tissue section of the spinal cord taken at 7 dpi was double labeled for KCa3.1 ( J ) and CD11b (Mac-1 antibody; K ). Note the absence of double labeling of the Mac-1 + macrophages ( L ). Scale bars, 50 μm.

    Techniques Used: Labeling, Staining

    KCNN4 mRNA and KCa3.1 immunoreactivity are expressed in ex vivo macrophages and in primary cultures of macrophages/microglia. A–C , Representative images of double labeling for CD11b (Mac-1 antibody) ( A ) and KCa3.1 ( B ) of macrophages isolated from spinal cord 7 d after SCI; the merged image ( C ) includes nuclear DAPI staining. Note that the large, round Mac-1 + macrophage shows cytoplasmic labeling for KCa3.1. Scale bar: (in C ) A–C , 20 μm. D–F , Representative images of unstimulated cultured mouse microglia double labeled with tomato lectin ( D ) and anti-KCa3.1 antibody ( E ); the merged image ( F ) includes DAPI-stained nuclei. Scale bar: (in F ) D–F , 10 μm. G , RT-PCR analysis shows mRNA expression of KCNN4 in primary cultures of BMDMs and cultured microglia, with and without activation by LPS treatment. H , Note the much higher KCNN4 expression in BMDMs than in microglia. Values are mean ± SEM, n = 3.
    Figure Legend Snippet: KCNN4 mRNA and KCa3.1 immunoreactivity are expressed in ex vivo macrophages and in primary cultures of macrophages/microglia. A–C , Representative images of double labeling for CD11b (Mac-1 antibody) ( A ) and KCa3.1 ( B ) of macrophages isolated from spinal cord 7 d after SCI; the merged image ( C ) includes nuclear DAPI staining. Note that the large, round Mac-1 + macrophage shows cytoplasmic labeling for KCa3.1. Scale bar: (in C ) A–C , 20 μm. D–F , Representative images of unstimulated cultured mouse microglia double labeled with tomato lectin ( D ) and anti-KCa3.1 antibody ( E ); the merged image ( F ) includes DAPI-stained nuclei. Scale bar: (in F ) D–F , 10 μm. G , RT-PCR analysis shows mRNA expression of KCNN4 in primary cultures of BMDMs and cultured microglia, with and without activation by LPS treatment. H , Note the much higher KCNN4 expression in BMDMs than in microglia. Values are mean ± SEM, n = 3.

    Techniques Used: Ex Vivo, Labeling, Isolation, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Activation Assay

    KCa3.1 is expressed in neurons and some oligodendrocytes in the uninjured mouse spinal cord. A–C , Double labeling with antibodies against the neuronal marker NeuN ( A ) and the KCa3.1 channel ( B ) shows that the cell bodies of large neurons in the ventral horn express KCa3.1 ( C ). D–F , Double labeling with antibodies against the oligodendrocyte marker CC1 ( D ) and the KCa3.1 channel ( E ) shows KCa3.1 expression in the cell body and on the processes of some oligodendrocytes in the ventral white matter ( F ). This staining pattern was unchanged after SCI (data not shown). Scale bars, 50 μm.
    Figure Legend Snippet: KCa3.1 is expressed in neurons and some oligodendrocytes in the uninjured mouse spinal cord. A–C , Double labeling with antibodies against the neuronal marker NeuN ( A ) and the KCa3.1 channel ( B ) shows that the cell bodies of large neurons in the ventral horn express KCa3.1 ( C ). D–F , Double labeling with antibodies against the oligodendrocyte marker CC1 ( D ) and the KCa3.1 channel ( E ) shows KCa3.1 expression in the cell body and on the processes of some oligodendrocytes in the ventral white matter ( F ). This staining pattern was unchanged after SCI (data not shown). Scale bars, 50 μm.

    Techniques Used: Labeling, Marker, Expressing, Staining

    KCNN4 /KCa3.1 expression increases after SCI. A , B , RT-PCR analysis of KCNN4 mRNA levels in spinal cord from uninjured (ui) mice and 1, 3, 5, 7, 14, and 21 dpi mice. PPIA was used as a loading control. A representative example is shown in A . For the quantification in B , data were first normalized to PPIA and then expressed as fold change (mean ± SD; 3 experiments, n = 3) compared with the uninjured spinal cord (horizontal line). Significant increases in KCNN4 expression are indicated as follows: * p
    Figure Legend Snippet: KCNN4 /KCa3.1 expression increases after SCI. A , B , RT-PCR analysis of KCNN4 mRNA levels in spinal cord from uninjured (ui) mice and 1, 3, 5, 7, 14, and 21 dpi mice. PPIA was used as a loading control. A representative example is shown in A . For the quantification in B , data were first normalized to PPIA and then expressed as fold change (mean ± SD; 3 experiments, n = 3) compared with the uninjured spinal cord (horizontal line). Significant increases in KCNN4 expression are indicated as follows: * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay

    9) Product Images from "KCa3.1 constitutes a pharmacological target for astrogliosis associated with Alzheimer’s disease"

    Article Title: KCa3.1 constitutes a pharmacological target for astrogliosis associated with Alzheimer’s disease

    Journal: Molecular and cellular neurosciences

    doi: 10.1016/j.mcn.2016.08.008

    KCa3.1 deletion rescued memory deficits after hippocampal injection of Aβ oligomers. (A) Escape latency; (B) Percentage of time spent in the target quadrant where the escape platform had been located for groups of WT (n = 8–10) and KCa3.1 −/− (n = 8–10) mice after intrahippocampal injection of Aβ oligomers. Data represent mean ± SEM. # p
    Figure Legend Snippet: KCa3.1 deletion rescued memory deficits after hippocampal injection of Aβ oligomers. (A) Escape latency; (B) Percentage of time spent in the target quadrant where the escape platform had been located for groups of WT (n = 8–10) and KCa3.1 −/− (n = 8–10) mice after intrahippocampal injection of Aβ oligomers. Data represent mean ± SEM. # p

    Techniques Used: Injection, Mouse Assay

    Blockade of KCa3.1 attenuated the activation of astrocytes and microglia in SAMP8 mice. Seven-month-old SAMP8 mice (n = 6–8 per group) were treated with vehicle or TRAM-34 (60 or 120 mg/kg, intraperitoneal) daily for 4 weeks with age-matched vehicle treated SAMR1 mice as normal controls. Brain sections were immunostained with anti-GFAP and anti-Iba-1 antibodies. (A) Representative images of GFAP-immunoreactive astrocytes from the hippocampal regions of SAMR1, SAMP8 or SAMP8 + TRAM-34 (120 mg/kg) groups. Scale bar: 50 μm. (B) Quantification of reactive astrocyte number/0.01 mm 2 in the hippocampus (n = 6–8). Data represent mean ± SEM. ## p
    Figure Legend Snippet: Blockade of KCa3.1 attenuated the activation of astrocytes and microglia in SAMP8 mice. Seven-month-old SAMP8 mice (n = 6–8 per group) were treated with vehicle or TRAM-34 (60 or 120 mg/kg, intraperitoneal) daily for 4 weeks with age-matched vehicle treated SAMR1 mice as normal controls. Brain sections were immunostained with anti-GFAP and anti-Iba-1 antibodies. (A) Representative images of GFAP-immunoreactive astrocytes from the hippocampal regions of SAMR1, SAMP8 or SAMP8 + TRAM-34 (120 mg/kg) groups. Scale bar: 50 μm. (B) Quantification of reactive astrocyte number/0.01 mm 2 in the hippocampus (n = 6–8). Data represent mean ± SEM. ## p

    Techniques Used: Activation Assay, Mouse Assay

    Up-regulation of KCa3.1 in reactive astrocytes and neurons of SAMP8 mouse brains. Double immunofluorescence staining of KCa3.1 (green) with GFAP (red), NeuN (red) or Iba1 (red) in brain sections of 7-month-old SAMR1 and SAMP8 mice. (A) Co-staining of KCa3.1 and GFAP in SAMR1 and SAMP8 mice; (B) Co-staining of KCa3.1 and NeuN in SAMR1 and SAMP8 mice; (C) Co-staining of KCa3.1 and Iba1 in SAMR1 and SAMP8 mice. Arrows indicate co-labeling of KCa3.1 and GFAP (A), KCa3.1 and NeuN (B) and KCa3.1 and Iba1 (C), views of which are enlarged in the adjacent panels. Nuclei were stained in blue with DAPI. Scale bar: 25 μm in A; Scale bar: 50 μm in B; Scale bar: 25 μm in C.
    Figure Legend Snippet: Up-regulation of KCa3.1 in reactive astrocytes and neurons of SAMP8 mouse brains. Double immunofluorescence staining of KCa3.1 (green) with GFAP (red), NeuN (red) or Iba1 (red) in brain sections of 7-month-old SAMR1 and SAMP8 mice. (A) Co-staining of KCa3.1 and GFAP in SAMR1 and SAMP8 mice; (B) Co-staining of KCa3.1 and NeuN in SAMR1 and SAMP8 mice; (C) Co-staining of KCa3.1 and Iba1 in SAMR1 and SAMP8 mice. Arrows indicate co-labeling of KCa3.1 and GFAP (A), KCa3.1 and NeuN (B) and KCa3.1 and Iba1 (C), views of which are enlarged in the adjacent panels. Nuclei were stained in blue with DAPI. Scale bar: 25 μm in A; Scale bar: 50 μm in B; Scale bar: 25 μm in C.

    Techniques Used: Double Immunofluorescence Staining, Mouse Assay, Staining, Labeling

    Involvement of KCa3.1 in Aβ oligomer induced reactive astrogliosis. (A) Representative western blot showing stimulation with 1 μM Aβ oligomer increased KCa3.1 and GFAP protein expression in cultured astrocytes in a time-dependent fashion. Quantification of western blot for KCa3.1 and GFAP expression in astrocytes after Aβ oligomer treatment (n = 3). Con: control. Data are presented as mean ± SEM. * p
    Figure Legend Snippet: Involvement of KCa3.1 in Aβ oligomer induced reactive astrogliosis. (A) Representative western blot showing stimulation with 1 μM Aβ oligomer increased KCa3.1 and GFAP protein expression in cultured astrocytes in a time-dependent fashion. Quantification of western blot for KCa3.1 and GFAP expression in astrocytes after Aβ oligomer treatment (n = 3). Con: control. Data are presented as mean ± SEM. * p

    Techniques Used: Western Blot, Expressing, Cell Culture

    Involvement of KCa3.1 in Aβ oligomer-induced intracellular calcium increase in astrocytes. Astrocytes were loaded with the Ca 2+ -sensitive dye Fluo-4 AM at 37 ˚C for 30 min and changes in [Ca 2+ ] i were monitored by confocal microscopy. 5 μM Aβ oligomer was added to astrocytes at 30 s with or without pre-treated TRAM-34 (1 μM) (A). Summary of data showing intracellular Ca 2+ (B) at 1 s, 180 s (peak of fluorescence) and 1000 s. * p
    Figure Legend Snippet: Involvement of KCa3.1 in Aβ oligomer-induced intracellular calcium increase in astrocytes. Astrocytes were loaded with the Ca 2+ -sensitive dye Fluo-4 AM at 37 ˚C for 30 min and changes in [Ca 2+ ] i were monitored by confocal microscopy. 5 μM Aβ oligomer was added to astrocytes at 30 s with or without pre-treated TRAM-34 (1 μM) (A). Summary of data showing intracellular Ca 2+ (B) at 1 s, 180 s (peak of fluorescence) and 1000 s. * p

    Techniques Used: Confocal Microscopy, Fluorescence

    Up-regulation of KCa3.1 in the brains of SAMP8 mice. (A) Western blot analysis of KCa3.1 protein expression in the cortex of 3-, 7- and 12-month-old SAMR1 and SAMP8 mice. β-actin was used as the loading control. (B) The quantification of KCa3.1 protein expression in SAMR1 and SAMP8 mice (n = 3 each group). * p
    Figure Legend Snippet: Up-regulation of KCa3.1 in the brains of SAMP8 mice. (A) Western blot analysis of KCa3.1 protein expression in the cortex of 3-, 7- and 12-month-old SAMR1 and SAMP8 mice. β-actin was used as the loading control. (B) The quantification of KCa3.1 protein expression in SAMR1 and SAMP8 mice (n = 3 each group). * p

    Techniques Used: Mouse Assay, Western Blot, Expressing

    Up-regulation of KCa3.1 in reactive glial cells and neurons of Alzheimer’s patient brains. Double immunofluorescence staining of KCa3.1 (green) with GFAP (red), NeuN (red) or Iba1 (red) in brain sections of control and AD patients. (A) Co-staining of KCa3.1 and GFAP in control human and AD patients; (B) Co-staining of KCa3.1 and NeuN in control human and AD patients; (C) Co-staining of KCa3.1 and Iba1 in control human and AD patients. Arrows indicate co-labeling of KCa3.1 and GFAP (A), the co-labeling of KCa3.1 and NeuN (B) and the co-labeling of KCa3.1 and Iba1 (C). Nuclei were stained in blue with DAPI. Scale bar: 25 μm.
    Figure Legend Snippet: Up-regulation of KCa3.1 in reactive glial cells and neurons of Alzheimer’s patient brains. Double immunofluorescence staining of KCa3.1 (green) with GFAP (red), NeuN (red) or Iba1 (red) in brain sections of control and AD patients. (A) Co-staining of KCa3.1 and GFAP in control human and AD patients; (B) Co-staining of KCa3.1 and NeuN in control human and AD patients; (C) Co-staining of KCa3.1 and Iba1 in control human and AD patients. Arrows indicate co-labeling of KCa3.1 and GFAP (A), the co-labeling of KCa3.1 and NeuN (B) and the co-labeling of KCa3.1 and Iba1 (C). Nuclei were stained in blue with DAPI. Scale bar: 25 μm.

    Techniques Used: Double Immunofluorescence Staining, Staining, Labeling

    Blockade of KCa3.1 rescued memory deficits in SAMP8 mice using Morris water maze test. Seven-month-old SAMP8 mice (n = 8–10 per group) were treated with vehicle or TRAM-34 (60 or 120 mg/kg, intraperitoneal) daily for 4 weeks with the age-matched vehicle treated SAMR1 mice as normal controls, and three-month-old SAMP8 mice as negative controls. (A) Average daily escape latency; (B) Percentage of time spent in the target quadrant where the escape platform was located. Data represent mean ± SEM. ## p
    Figure Legend Snippet: Blockade of KCa3.1 rescued memory deficits in SAMP8 mice using Morris water maze test. Seven-month-old SAMP8 mice (n = 8–10 per group) were treated with vehicle or TRAM-34 (60 or 120 mg/kg, intraperitoneal) daily for 4 weeks with the age-matched vehicle treated SAMR1 mice as normal controls, and three-month-old SAMP8 mice as negative controls. (A) Average daily escape latency; (B) Percentage of time spent in the target quadrant where the escape platform was located. Data represent mean ± SEM. ## p

    Techniques Used: Mouse Assay

    KCa3.1 deletion attenuated gliosis in response to hippocampal injection of Aβ oligomers. (A) Representative images of GFAP-immunoreactive astrocytes from the hippocampal regions of WT or KCa3.1 KO mice at 14 days after Aβ oligomer injection. Scale bar: 50 μm. (B) Quantification of reactive astrocyte number/0.01 mm 2 in the hippocampus (n = 6–8). (C) Iba-1 immunoreactivity demonstrated active microglia from the hippocampal regions of WT or KCa3.1 KO mice at 14 days after Aβ oligomer intrahippocampus injection. Scale bar: 25 μm. (D) Quantification of activated microglia number/0.01 mm 2 in the hippocampus (n = 6–8). (E) NeuN immunoreactivity demonstrated neurons from the hippocampal regions of WT or KCa3.1 KO mice at 14 days after Aβ oligomer injection. Scale bar: 25 μm. (F) Quantification of activated microglia number/0.01 mm 2 in the hippocampus (n = 6–8). Data represent mean ± SEM. ** p
    Figure Legend Snippet: KCa3.1 deletion attenuated gliosis in response to hippocampal injection of Aβ oligomers. (A) Representative images of GFAP-immunoreactive astrocytes from the hippocampal regions of WT or KCa3.1 KO mice at 14 days after Aβ oligomer injection. Scale bar: 50 μm. (B) Quantification of reactive astrocyte number/0.01 mm 2 in the hippocampus (n = 6–8). (C) Iba-1 immunoreactivity demonstrated active microglia from the hippocampal regions of WT or KCa3.1 KO mice at 14 days after Aβ oligomer intrahippocampus injection. Scale bar: 25 μm. (D) Quantification of activated microglia number/0.01 mm 2 in the hippocampus (n = 6–8). (E) NeuN immunoreactivity demonstrated neurons from the hippocampal regions of WT or KCa3.1 KO mice at 14 days after Aβ oligomer injection. Scale bar: 25 μm. (F) Quantification of activated microglia number/0.01 mm 2 in the hippocampus (n = 6–8). Data represent mean ± SEM. ** p

    Techniques Used: Injection, Mouse Assay

    10) Product Images from "Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling"

    Article Title: Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1351-x

    AKT modulation is crucial for KCa3.1-mediated ER stress in astrocytes. a Representative blots of p-AKT and total AKT from the hippocampi of postmortem human AD patients and age-matched controls. b Data are presented as the mean ± SEM ( n = 3–5). The OD value of p-AKT was normalized to that of AKT. ** p
    Figure Legend Snippet: AKT modulation is crucial for KCa3.1-mediated ER stress in astrocytes. a Representative blots of p-AKT and total AKT from the hippocampi of postmortem human AD patients and age-matched controls. b Data are presented as the mean ± SEM ( n = 3–5). The OD value of p-AKT was normalized to that of AKT. ** p

    Techniques Used:

    Elimination of KCa3.1 in APP/PS1 mice rescues spatial memory deficits in the MWM test. MWM testing of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice was performed as described in the “ Materials and methods ” section. a Escape latency. b Number of crossing the target quadrant by each group during the probe trials (no platform). c Percentage of swimming time spent in the target quadrant by each group during the probe trials (no platform). d Percentage of swimming distance spent in the target quadrant by each group during the probe trials (no platform). Data are presented as the mean ± SEM ( n = 10–12). # p
    Figure Legend Snippet: Elimination of KCa3.1 in APP/PS1 mice rescues spatial memory deficits in the MWM test. MWM testing of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice was performed as described in the “ Materials and methods ” section. a Escape latency. b Number of crossing the target quadrant by each group during the probe trials (no platform). c Percentage of swimming time spent in the target quadrant by each group during the probe trials (no platform). d Percentage of swimming distance spent in the target quadrant by each group during the probe trials (no platform). Data are presented as the mean ± SEM ( n = 10–12). # p

    Techniques Used: Mouse Assay

    Neuronal loss is rescued in brains of KCa3.1 −/− /APP/PS1 mice. a Immunofluorescence analysis of NeuN levels in the hippocampi of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b Quantification of neuron number/0.01 mm 2 in the hippocampus ( n = 6). Data are presented as the mean ± SEM. * p
    Figure Legend Snippet: Neuronal loss is rescued in brains of KCa3.1 −/− /APP/PS1 mice. a Immunofluorescence analysis of NeuN levels in the hippocampi of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b Quantification of neuron number/0.01 mm 2 in the hippocampus ( n = 6). Data are presented as the mean ± SEM. * p

    Techniques Used: Mouse Assay, Immunofluorescence

    Decreased neuroinflammation in brains of KCa3.1 −/− /APP/PS1 mice. a Levels of activated microglia in CA1 areas of the mouse hippocampus were analyzed by immunostaining of the microglia marker Iba1. b Quantification of activated microglia number/0.01 mm 2 in the hippocampus ( n = 3). Data are presented as the mean ± SEM. * p
    Figure Legend Snippet: Decreased neuroinflammation in brains of KCa3.1 −/− /APP/PS1 mice. a Levels of activated microglia in CA1 areas of the mouse hippocampus were analyzed by immunostaining of the microglia marker Iba1. b Quantification of activated microglia number/0.01 mm 2 in the hippocampus ( n = 3). Data are presented as the mean ± SEM. * p

    Techniques Used: Mouse Assay, Immunostaining, Marker

    KCa3.1 upregulation in Aβ-induced RA and the brains of AD patients. a Primary astrocytes were stimulated with 5 μM Aβ and lysates were subjected to Western blot analysis with antibodies against KCa3.1, Orai1, and STIM1. β-actin was used to confirm equal loading. b Data are presented as the mean ± SEM ( n = 5). The OD values of KCa3.1, Orai1, and STIM1 were normalized to that of β-actin. * p
    Figure Legend Snippet: KCa3.1 upregulation in Aβ-induced RA and the brains of AD patients. a Primary astrocytes were stimulated with 5 μM Aβ and lysates were subjected to Western blot analysis with antibodies against KCa3.1, Orai1, and STIM1. β-actin was used to confirm equal loading. b Data are presented as the mean ± SEM ( n = 5). The OD values of KCa3.1, Orai1, and STIM1 were normalized to that of β-actin. * p

    Techniques Used: Western Blot

    KCa3.1 contributes to increased ER stress in APP/PS1 mice. a Western blot analysis of GRP78 and CHOP protein levels in hippocampal extracts of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b , c Data are presented as the mean ± SEM ( n = 3–5). The OD values of GRP78 ( b ) and CHOP ( c ) were normalized to that of β-actin. # p
    Figure Legend Snippet: KCa3.1 contributes to increased ER stress in APP/PS1 mice. a Western blot analysis of GRP78 and CHOP protein levels in hippocampal extracts of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b , c Data are presented as the mean ± SEM ( n = 3–5). The OD values of GRP78 ( b ) and CHOP ( c ) were normalized to that of β-actin. # p

    Techniques Used: Mouse Assay, Western Blot

    KCa3.1 involved in astrocytes SOCE and ER stress. a Primary cultured astrocytes were treated with 5 μM Aβ for 12 h with or without pretreatment of the KCa3.1 blocker TRAM-34 (1 μM). Fluorescence intensities of [Ca 2+ ] i are shown. Fluorescence intensity was measured in the presence of 1 μM Tg with or without 2 mM Ca 2+ . b Data are presented as the mean ± SEM ( n = 10). # p
    Figure Legend Snippet: KCa3.1 involved in astrocytes SOCE and ER stress. a Primary cultured astrocytes were treated with 5 μM Aβ for 12 h with or without pretreatment of the KCa3.1 blocker TRAM-34 (1 μM). Fluorescence intensities of [Ca 2+ ] i are shown. Fluorescence intensity was measured in the presence of 1 μM Tg with or without 2 mM Ca 2+ . b Data are presented as the mean ± SEM ( n = 10). # p

    Techniques Used: Cell Culture, Fluorescence

    11) Product Images from "Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury"

    Article Title: Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury

    Journal: Acta Pharmaceutica Sinica. B

    doi: 10.1016/j.apsb.2020.05.002

    Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P
    Figure Legend Snippet: Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P

    Techniques Used: Mouse Assay, Expressing, Immunostaining, Staining, Marker, MANN-WHITNEY

    A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.
    Figure Legend Snippet: A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.

    Techniques Used: Binding Assay

    12) Product Images from "The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease"

    Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-019-1682-2

    AKT modulation is crucial for KCa3.1-mediated ER stress in microglia. a , b Representative blots of p-AKT and total AKT in SNpc from a WT, WT+MPTP, KCa3.1 −/− , KCa3.1 −/− +MPTP group mice and from b control, MPTP, MPTP+Se, Se group mice. Data are presented as the mean ± SEM ( n = 3–5). Western blot was repeated three times and showed similar results. The OD value of p-AKT was normalized to that of AKT. * p
    Figure Legend Snippet: AKT modulation is crucial for KCa3.1-mediated ER stress in microglia. a , b Representative blots of p-AKT and total AKT in SNpc from a WT, WT+MPTP, KCa3.1 −/− , KCa3.1 −/− +MPTP group mice and from b control, MPTP, MPTP+Se, Se group mice. Data are presented as the mean ± SEM ( n = 3–5). Western blot was repeated three times and showed similar results. The OD value of p-AKT was normalized to that of AKT. * p

    Techniques Used: Mouse Assay, Western Blot

    KCa3.1 involved in microglia SOCE and ER stress. a , b Representative images of GRP78, p-PERK, and p-eIF2α in KCa3.1 −/− microglia, responses to 500 μM MPP + ( a ) or 1 μM Tg ( b ) vs. WT cells. Mean values of GRP78, p-PERK, and p-eIF2α relative to β-actin. Data are presented as the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results. * p
    Figure Legend Snippet: KCa3.1 involved in microglia SOCE and ER stress. a , b Representative images of GRP78, p-PERK, and p-eIF2α in KCa3.1 −/− microglia, responses to 500 μM MPP + ( a ) or 1 μM Tg ( b ) vs. WT cells. Mean values of GRP78, p-PERK, and p-eIF2α relative to β-actin. Data are presented as the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results. * p

    Techniques Used: Western Blot

    Upregulation of KCa3.1 channels and Iba1 in the brains of PD mouse model. a Western blot analysis of SNpc lysates from control and MPTP-induced PD mouse model analyzed by antibodies to TH, GFAP, Iba1, and KCa3.1. Data represent the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results; * p
    Figure Legend Snippet: Upregulation of KCa3.1 channels and Iba1 in the brains of PD mouse model. a Western blot analysis of SNpc lysates from control and MPTP-induced PD mouse model analyzed by antibodies to TH, GFAP, Iba1, and KCa3.1. Data represent the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results; * p

    Techniques Used: Western Blot

    Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced loss of DA neurons. a – g WT or KCa3.1 −/− mice received sequential intraperitoneal injections of MPTP (20 mg/kg) with or without senicapoc (100 mg/kg, once daily, p.o.) treatment for 5 days as described in the “ Material and methods ” section. Open field test ( b – e ) and the rotarod test ( f , g ) for bradykinesia were performed. Behavioral tests for MPTP-induced bradykinesia were conducted on the indicated days. Data are presented as mean ± SEM ( n = 10–15). b – e ** p
    Figure Legend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced loss of DA neurons. a – g WT or KCa3.1 −/− mice received sequential intraperitoneal injections of MPTP (20 mg/kg) with or without senicapoc (100 mg/kg, once daily, p.o.) treatment for 5 days as described in the “ Material and methods ” section. Open field test ( b – e ) and the rotarod test ( f , g ) for bradykinesia were performed. Behavioral tests for MPTP-induced bradykinesia were conducted on the indicated days. Data are presented as mean ± SEM ( n = 10–15). b – e ** p

    Techniques Used: Mouse Assay

    Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuated MPTP-induced ER stress. a , d Western blot analysis of GRP78 and CHOP protein levels in SNpc. b , c , e , f Data are presented as the mean ± SEM ( n = 5–6). Western blot was repeated three times and showed similar results. # p
    Figure Legend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuated MPTP-induced ER stress. a , d Western blot analysis of GRP78 and CHOP protein levels in SNpc. b , c , e , f Data are presented as the mean ± SEM ( n = 5–6). Western blot was repeated three times and showed similar results. # p

    Techniques Used: Western Blot

    Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced microgliosis. a , c Immunostaining for Iba1 in SNpc. Bar 50 μM. Quantitative analysis of Iba1 + cells in SNpc. Data are presented as mean ± SEM ( n = 5–8). * p
    Figure Legend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced microgliosis. a , c Immunostaining for Iba1 in SNpc. Bar 50 μM. Quantitative analysis of Iba1 + cells in SNpc. Data are presented as mean ± SEM ( n = 5–8). * p

    Techniques Used: Immunostaining

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    Alomone Labs mouse anti kca3 1
    Role of TRPV4 and <t>KCa3.1</t> in alteration of membrane potential and Ca 2+ entry in astrocytes following OGD. a–f Changes in membrane potential in response to activation of KCa3.1 channels and TRPV4 channels in astrocytes exposed to OGD 1 h. a , b 1-EBIO was added to WT astrocytes and membrane potential measured with or without OGD or HC 067047. Data are presented as means ± SEM. n = 10–20. *** p
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    Role of TRPV4 and KCa3.1 in alteration of membrane potential and Ca 2+ entry in astrocytes following OGD. a–f Changes in membrane potential in response to activation of KCa3.1 channels and TRPV4 channels in astrocytes exposed to OGD 1 h. a , b 1-EBIO was added to WT astrocytes and membrane potential measured with or without OGD or HC 067047. Data are presented as means ± SEM. n = 10–20. *** p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke

    doi: 10.1186/s12974-017-0973-8

    Figure Lengend Snippet: Role of TRPV4 and KCa3.1 in alteration of membrane potential and Ca 2+ entry in astrocytes following OGD. a–f Changes in membrane potential in response to activation of KCa3.1 channels and TRPV4 channels in astrocytes exposed to OGD 1 h. a , b 1-EBIO was added to WT astrocytes and membrane potential measured with or without OGD or HC 067047. Data are presented as means ± SEM. n = 10–20. *** p

    Article Snippet: Sections and cells were incubated at 4 °C overnight with primary antibodies: mouse anti-KCa3.1 (1:100; Alomone Labs), rabbit anti-GFAP (1:500; Dako); rabbit anti-Iba1 (1:500; Abcam); rabbit anti-NeuN antibody (1:500; Millipore), rabbit anti-TRPV4 (1:200; Alomone Labs).

    Techniques: Activation Assay

    KCa3.1 and TRPV4 co-localized in primary cultured astrocytes and mouse brain cortex. Double immunofluorescence images of KCa3.1 (green) and TRPV4 (red) in normal mouse brains ( a , b ), and primary cultured astrocytes ( c , d ). Note the strong co-localization indicated by merge yellow fluorescence, quantification of the co-localization observed in experiments as shown in g. ( e ) The histograms represent the ratio of the mean Pearson correlation coefficient calculated from the co-labeling in a number of samples, as indicated above the bar. Scale bar: 25 μm

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke

    doi: 10.1186/s12974-017-0973-8

    Figure Lengend Snippet: KCa3.1 and TRPV4 co-localized in primary cultured astrocytes and mouse brain cortex. Double immunofluorescence images of KCa3.1 (green) and TRPV4 (red) in normal mouse brains ( a , b ), and primary cultured astrocytes ( c , d ). Note the strong co-localization indicated by merge yellow fluorescence, quantification of the co-localization observed in experiments as shown in g. ( e ) The histograms represent the ratio of the mean Pearson correlation coefficient calculated from the co-labeling in a number of samples, as indicated above the bar. Scale bar: 25 μm

    Article Snippet: Sections and cells were incubated at 4 °C overnight with primary antibodies: mouse anti-KCa3.1 (1:100; Alomone Labs), rabbit anti-GFAP (1:500; Dako); rabbit anti-Iba1 (1:500; Abcam); rabbit anti-NeuN antibody (1:500; Millipore), rabbit anti-TRPV4 (1:200; Alomone Labs).

    Techniques: Cell Culture, Immunofluorescence, Fluorescence, Labeling

    Involvement of KCa3.1 in OGD-induced reactive astrogliosis. a , b Representative western blot showing GFAP expression in cultured astrocytes treated with OGD for 4 h in the presence of 1 μM TRAM-34 and 10 μM HC 067047. Quantification of western blot for GFAP expression ( n = 3). Data are presented as means ± SEM. # p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke

    doi: 10.1186/s12974-017-0973-8

    Figure Lengend Snippet: Involvement of KCa3.1 in OGD-induced reactive astrogliosis. a , b Representative western blot showing GFAP expression in cultured astrocytes treated with OGD for 4 h in the presence of 1 μM TRAM-34 and 10 μM HC 067047. Quantification of western blot for GFAP expression ( n = 3). Data are presented as means ± SEM. # p

    Article Snippet: Sections and cells were incubated at 4 °C overnight with primary antibodies: mouse anti-KCa3.1 (1:100; Alomone Labs), rabbit anti-GFAP (1:500; Dako); rabbit anti-Iba1 (1:500; Abcam); rabbit anti-NeuN antibody (1:500; Millipore), rabbit anti-TRPV4 (1:200; Alomone Labs).

    Techniques: Western Blot, Expressing, Cell Culture

    Upregulation of KCa3.1 channels and GFAP in mouse brains following pMCAO. a , b Western blot analysis of lysates from 10-week-old male WT mice following 1, 3, 6, or 12 h of pMCAO analyzed by antibodies to KCa3.1 ( a ) and GFAP ( b ). Data represent the means ± SEM of KCa3.1 and GFAP density normalized to β-actin values for n = 3. * p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke

    doi: 10.1186/s12974-017-0973-8

    Figure Lengend Snippet: Upregulation of KCa3.1 channels and GFAP in mouse brains following pMCAO. a , b Western blot analysis of lysates from 10-week-old male WT mice following 1, 3, 6, or 12 h of pMCAO analyzed by antibodies to KCa3.1 ( a ) and GFAP ( b ). Data represent the means ± SEM of KCa3.1 and GFAP density normalized to β-actin values for n = 3. * p

    Article Snippet: Sections and cells were incubated at 4 °C overnight with primary antibodies: mouse anti-KCa3.1 (1:100; Alomone Labs), rabbit anti-GFAP (1:500; Dako); rabbit anti-Iba1 (1:500; Abcam); rabbit anti-NeuN antibody (1:500; Millipore), rabbit anti-TRPV4 (1:200; Alomone Labs).

    Techniques: Western Blot, Mouse Assay

    KCa3.1 deficiency reduces infarction volume and improves of neurological conditions. Focal cerebral ischemia was induced by pMCAO. a , c , and e Representative TTC staining of five corresponding coronal brain sections of a 10-week-old male WT mouse and a 10 week-old male KCa3.1 −/− mouse after 3 h ( a ), 6 h ( c ), and 24 h ( e ) of pMCAO. b , d , and f . Quantitative analysis of infarction volume in a, c, and e, respectively. Data are presented as means ± SEM. n = 6. * p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke

    doi: 10.1186/s12974-017-0973-8

    Figure Lengend Snippet: KCa3.1 deficiency reduces infarction volume and improves of neurological conditions. Focal cerebral ischemia was induced by pMCAO. a , c , and e Representative TTC staining of five corresponding coronal brain sections of a 10-week-old male WT mouse and a 10 week-old male KCa3.1 −/− mouse after 3 h ( a ), 6 h ( c ), and 24 h ( e ) of pMCAO. b , d , and f . Quantitative analysis of infarction volume in a, c, and e, respectively. Data are presented as means ± SEM. n = 6. * p

    Article Snippet: Sections and cells were incubated at 4 °C overnight with primary antibodies: mouse anti-KCa3.1 (1:100; Alomone Labs), rabbit anti-GFAP (1:500; Dako); rabbit anti-Iba1 (1:500; Abcam); rabbit anti-NeuN antibody (1:500; Millipore), rabbit anti-TRPV4 (1:200; Alomone Labs).

    Techniques: Staining

    Upregulation of KCa3.1, GFAP, and TRPV4 channels following OGD in cultured astrocytes. Western blot analysis of ( a ) KCa3.1, ( b ) GFAP, and ( c ) TRPV4 expression after OGD-treatment for 0, 1, 3, 4, 6, 12 h. Data represent the means ± SEM of KCa3.1, GFAP, and TRPV4 density normalized to β-actin values for n = 3 cultures. * p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke

    doi: 10.1186/s12974-017-0973-8

    Figure Lengend Snippet: Upregulation of KCa3.1, GFAP, and TRPV4 channels following OGD in cultured astrocytes. Western blot analysis of ( a ) KCa3.1, ( b ) GFAP, and ( c ) TRPV4 expression after OGD-treatment for 0, 1, 3, 4, 6, 12 h. Data represent the means ± SEM of KCa3.1, GFAP, and TRPV4 density normalized to β-actin values for n = 3 cultures. * p

    Article Snippet: Sections and cells were incubated at 4 °C overnight with primary antibodies: mouse anti-KCa3.1 (1:100; Alomone Labs), rabbit anti-GFAP (1:500; Dako); rabbit anti-Iba1 (1:500; Abcam); rabbit anti-NeuN antibody (1:500; Millipore), rabbit anti-TRPV4 (1:200; Alomone Labs).

    Techniques: Cell Culture, Western Blot, Expressing

    Decreased glial activation and neuronal loss in brains of KCa3.1 deletion mice following pMCAO. Reactive astrocytes ( a ), activated microglia ( b ), and neurons ( c ) from the hippocampal CA1 regions of WT or KCa3.1 −/− mice at 6 h after pMCAO were visualized by GFAP, Iba1, and NeuN immunostaining, respectively. At least four coronal slices from each mouse brain and at least three brains of each genotype were used for immunostaining and counting. n = 4 per group. Scale bar: 75 μm. Data represent means ± SEM. * p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke

    doi: 10.1186/s12974-017-0973-8

    Figure Lengend Snippet: Decreased glial activation and neuronal loss in brains of KCa3.1 deletion mice following pMCAO. Reactive astrocytes ( a ), activated microglia ( b ), and neurons ( c ) from the hippocampal CA1 regions of WT or KCa3.1 −/− mice at 6 h after pMCAO were visualized by GFAP, Iba1, and NeuN immunostaining, respectively. At least four coronal slices from each mouse brain and at least three brains of each genotype were used for immunostaining and counting. n = 4 per group. Scale bar: 75 μm. Data represent means ± SEM. * p

    Article Snippet: Sections and cells were incubated at 4 °C overnight with primary antibodies: mouse anti-KCa3.1 (1:100; Alomone Labs), rabbit anti-GFAP (1:500; Dako); rabbit anti-Iba1 (1:500; Abcam); rabbit anti-NeuN antibody (1:500; Millipore), rabbit anti-TRPV4 (1:200; Alomone Labs).

    Techniques: Activation Assay, Mouse Assay, Immunostaining

    AKT modulation is crucial for KCa3.1-mediated ER stress in microglia. a , b Representative blots of p-AKT and total AKT in SNpc from a WT, WT+MPTP, KCa3.1 −/− , KCa3.1 −/− +MPTP group mice and from b control, MPTP, MPTP+Se, Se group mice. Data are presented as the mean ± SEM ( n = 3–5). Western blot was repeated three times and showed similar results. The OD value of p-AKT was normalized to that of AKT. * p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

    doi: 10.1186/s12974-019-1682-2

    Figure Lengend Snippet: AKT modulation is crucial for KCa3.1-mediated ER stress in microglia. a , b Representative blots of p-AKT and total AKT in SNpc from a WT, WT+MPTP, KCa3.1 −/− , KCa3.1 −/− +MPTP group mice and from b control, MPTP, MPTP+Se, Se group mice. Data are presented as the mean ± SEM ( n = 3–5). Western blot was repeated three times and showed similar results. The OD value of p-AKT was normalized to that of AKT. * p

    Article Snippet: The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel).

    Techniques: Mouse Assay, Western Blot

    KCa3.1 involved in microglia SOCE and ER stress. a , b Representative images of GRP78, p-PERK, and p-eIF2α in KCa3.1 −/− microglia, responses to 500 μM MPP + ( a ) or 1 μM Tg ( b ) vs. WT cells. Mean values of GRP78, p-PERK, and p-eIF2α relative to β-actin. Data are presented as the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results. * p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

    doi: 10.1186/s12974-019-1682-2

    Figure Lengend Snippet: KCa3.1 involved in microglia SOCE and ER stress. a , b Representative images of GRP78, p-PERK, and p-eIF2α in KCa3.1 −/− microglia, responses to 500 μM MPP + ( a ) or 1 μM Tg ( b ) vs. WT cells. Mean values of GRP78, p-PERK, and p-eIF2α relative to β-actin. Data are presented as the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results. * p

    Article Snippet: The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel).

    Techniques: Western Blot

    Upregulation of KCa3.1 channels and Iba1 in the brains of PD mouse model. a Western blot analysis of SNpc lysates from control and MPTP-induced PD mouse model analyzed by antibodies to TH, GFAP, Iba1, and KCa3.1. Data represent the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results; * p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

    doi: 10.1186/s12974-019-1682-2

    Figure Lengend Snippet: Upregulation of KCa3.1 channels and Iba1 in the brains of PD mouse model. a Western blot analysis of SNpc lysates from control and MPTP-induced PD mouse model analyzed by antibodies to TH, GFAP, Iba1, and KCa3.1. Data represent the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results; * p

    Article Snippet: The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel).

    Techniques: Western Blot

    Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced loss of DA neurons. a – g WT or KCa3.1 −/− mice received sequential intraperitoneal injections of MPTP (20 mg/kg) with or without senicapoc (100 mg/kg, once daily, p.o.) treatment for 5 days as described in the “ Material and methods ” section. Open field test ( b – e ) and the rotarod test ( f , g ) for bradykinesia were performed. Behavioral tests for MPTP-induced bradykinesia were conducted on the indicated days. Data are presented as mean ± SEM ( n = 10–15). b – e ** p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

    doi: 10.1186/s12974-019-1682-2

    Figure Lengend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced loss of DA neurons. a – g WT or KCa3.1 −/− mice received sequential intraperitoneal injections of MPTP (20 mg/kg) with or without senicapoc (100 mg/kg, once daily, p.o.) treatment for 5 days as described in the “ Material and methods ” section. Open field test ( b – e ) and the rotarod test ( f , g ) for bradykinesia were performed. Behavioral tests for MPTP-induced bradykinesia were conducted on the indicated days. Data are presented as mean ± SEM ( n = 10–15). b – e ** p

    Article Snippet: The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel).

    Techniques: Mouse Assay

    Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuated MPTP-induced ER stress. a , d Western blot analysis of GRP78 and CHOP protein levels in SNpc. b , c , e , f Data are presented as the mean ± SEM ( n = 5–6). Western blot was repeated three times and showed similar results. # p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

    doi: 10.1186/s12974-019-1682-2

    Figure Lengend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuated MPTP-induced ER stress. a , d Western blot analysis of GRP78 and CHOP protein levels in SNpc. b , c , e , f Data are presented as the mean ± SEM ( n = 5–6). Western blot was repeated three times and showed similar results. # p

    Article Snippet: The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel).

    Techniques: Western Blot

    Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced microgliosis. a , c Immunostaining for Iba1 in SNpc. Bar 50 μM. Quantitative analysis of Iba1 + cells in SNpc. Data are presented as mean ± SEM ( n = 5–8). * p

    Journal: Journal of Neuroinflammation

    Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

    doi: 10.1186/s12974-019-1682-2

    Figure Lengend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced microgliosis. a , c Immunostaining for Iba1 in SNpc. Bar 50 μM. Quantitative analysis of Iba1 + cells in SNpc. Data are presented as mean ± SEM ( n = 5–8). * p

    Article Snippet: The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel).

    Techniques: Immunostaining

    AKT modulation is crucial for KCa3.1-mediated ER stress in astrocytes. a Representative blots of p-AKT and total AKT from the hippocampi of postmortem human AD patients and age-matched controls. b Data are presented as the mean ± SEM ( n = 3–5). The OD value of p-AKT was normalized to that of AKT. ** p

    Journal: Journal of Neuroinflammation

    Article Title: Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling

    doi: 10.1186/s12974-018-1351-x

    Figure Lengend Snippet: AKT modulation is crucial for KCa3.1-mediated ER stress in astrocytes. a Representative blots of p-AKT and total AKT from the hippocampi of postmortem human AD patients and age-matched controls. b Data are presented as the mean ± SEM ( n = 3–5). The OD value of p-AKT was normalized to that of AKT. ** p

    Article Snippet: The brain sections were then incubated with the following primary antibodies: rabbit anti-GFAP (1:1000; Dako, Glostrup, Denmark), mouse anti-GFAP (1:200; Merck Millipore, Burlington, MA, USA), rabbit anti-NeuN (1:100; Merck Millipore, Burlington, MA, USA), rabbit anti-Iba1 (1:500; Wako Pure Chemical Industries, Ltd., Osaka, Japan), mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel), rabbit anti-GRP78 (1:100; Cell Signaling Technology, Inc., Beverly, MA, USA), mouse anti-NG2 (Sigma-Aldrich Corporation, St. Louis, MO, USA), and rabbit anti-Orai1 (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques:

    Elimination of KCa3.1 in APP/PS1 mice rescues spatial memory deficits in the MWM test. MWM testing of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice was performed as described in the “ Materials and methods ” section. a Escape latency. b Number of crossing the target quadrant by each group during the probe trials (no platform). c Percentage of swimming time spent in the target quadrant by each group during the probe trials (no platform). d Percentage of swimming distance spent in the target quadrant by each group during the probe trials (no platform). Data are presented as the mean ± SEM ( n = 10–12). # p

    Journal: Journal of Neuroinflammation

    Article Title: Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling

    doi: 10.1186/s12974-018-1351-x

    Figure Lengend Snippet: Elimination of KCa3.1 in APP/PS1 mice rescues spatial memory deficits in the MWM test. MWM testing of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice was performed as described in the “ Materials and methods ” section. a Escape latency. b Number of crossing the target quadrant by each group during the probe trials (no platform). c Percentage of swimming time spent in the target quadrant by each group during the probe trials (no platform). d Percentage of swimming distance spent in the target quadrant by each group during the probe trials (no platform). Data are presented as the mean ± SEM ( n = 10–12). # p

    Article Snippet: The brain sections were then incubated with the following primary antibodies: rabbit anti-GFAP (1:1000; Dako, Glostrup, Denmark), mouse anti-GFAP (1:200; Merck Millipore, Burlington, MA, USA), rabbit anti-NeuN (1:100; Merck Millipore, Burlington, MA, USA), rabbit anti-Iba1 (1:500; Wako Pure Chemical Industries, Ltd., Osaka, Japan), mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel), rabbit anti-GRP78 (1:100; Cell Signaling Technology, Inc., Beverly, MA, USA), mouse anti-NG2 (Sigma-Aldrich Corporation, St. Louis, MO, USA), and rabbit anti-Orai1 (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Mouse Assay

    Neuronal loss is rescued in brains of KCa3.1 −/− /APP/PS1 mice. a Immunofluorescence analysis of NeuN levels in the hippocampi of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b Quantification of neuron number/0.01 mm 2 in the hippocampus ( n = 6). Data are presented as the mean ± SEM. * p

    Journal: Journal of Neuroinflammation

    Article Title: Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling

    doi: 10.1186/s12974-018-1351-x

    Figure Lengend Snippet: Neuronal loss is rescued in brains of KCa3.1 −/− /APP/PS1 mice. a Immunofluorescence analysis of NeuN levels in the hippocampi of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b Quantification of neuron number/0.01 mm 2 in the hippocampus ( n = 6). Data are presented as the mean ± SEM. * p

    Article Snippet: The brain sections were then incubated with the following primary antibodies: rabbit anti-GFAP (1:1000; Dako, Glostrup, Denmark), mouse anti-GFAP (1:200; Merck Millipore, Burlington, MA, USA), rabbit anti-NeuN (1:100; Merck Millipore, Burlington, MA, USA), rabbit anti-Iba1 (1:500; Wako Pure Chemical Industries, Ltd., Osaka, Japan), mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel), rabbit anti-GRP78 (1:100; Cell Signaling Technology, Inc., Beverly, MA, USA), mouse anti-NG2 (Sigma-Aldrich Corporation, St. Louis, MO, USA), and rabbit anti-Orai1 (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Mouse Assay, Immunofluorescence

    Decreased neuroinflammation in brains of KCa3.1 −/− /APP/PS1 mice. a Levels of activated microglia in CA1 areas of the mouse hippocampus were analyzed by immunostaining of the microglia marker Iba1. b Quantification of activated microglia number/0.01 mm 2 in the hippocampus ( n = 3). Data are presented as the mean ± SEM. * p

    Journal: Journal of Neuroinflammation

    Article Title: Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling

    doi: 10.1186/s12974-018-1351-x

    Figure Lengend Snippet: Decreased neuroinflammation in brains of KCa3.1 −/− /APP/PS1 mice. a Levels of activated microglia in CA1 areas of the mouse hippocampus were analyzed by immunostaining of the microglia marker Iba1. b Quantification of activated microglia number/0.01 mm 2 in the hippocampus ( n = 3). Data are presented as the mean ± SEM. * p

    Article Snippet: The brain sections were then incubated with the following primary antibodies: rabbit anti-GFAP (1:1000; Dako, Glostrup, Denmark), mouse anti-GFAP (1:200; Merck Millipore, Burlington, MA, USA), rabbit anti-NeuN (1:100; Merck Millipore, Burlington, MA, USA), rabbit anti-Iba1 (1:500; Wako Pure Chemical Industries, Ltd., Osaka, Japan), mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel), rabbit anti-GRP78 (1:100; Cell Signaling Technology, Inc., Beverly, MA, USA), mouse anti-NG2 (Sigma-Aldrich Corporation, St. Louis, MO, USA), and rabbit anti-Orai1 (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Mouse Assay, Immunostaining, Marker

    KCa3.1 upregulation in Aβ-induced RA and the brains of AD patients. a Primary astrocytes were stimulated with 5 μM Aβ and lysates were subjected to Western blot analysis with antibodies against KCa3.1, Orai1, and STIM1. β-actin was used to confirm equal loading. b Data are presented as the mean ± SEM ( n = 5). The OD values of KCa3.1, Orai1, and STIM1 were normalized to that of β-actin. * p

    Journal: Journal of Neuroinflammation

    Article Title: Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling

    doi: 10.1186/s12974-018-1351-x

    Figure Lengend Snippet: KCa3.1 upregulation in Aβ-induced RA and the brains of AD patients. a Primary astrocytes were stimulated with 5 μM Aβ and lysates were subjected to Western blot analysis with antibodies against KCa3.1, Orai1, and STIM1. β-actin was used to confirm equal loading. b Data are presented as the mean ± SEM ( n = 5). The OD values of KCa3.1, Orai1, and STIM1 were normalized to that of β-actin. * p

    Article Snippet: The brain sections were then incubated with the following primary antibodies: rabbit anti-GFAP (1:1000; Dako, Glostrup, Denmark), mouse anti-GFAP (1:200; Merck Millipore, Burlington, MA, USA), rabbit anti-NeuN (1:100; Merck Millipore, Burlington, MA, USA), rabbit anti-Iba1 (1:500; Wako Pure Chemical Industries, Ltd., Osaka, Japan), mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel), rabbit anti-GRP78 (1:100; Cell Signaling Technology, Inc., Beverly, MA, USA), mouse anti-NG2 (Sigma-Aldrich Corporation, St. Louis, MO, USA), and rabbit anti-Orai1 (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Western Blot

    KCa3.1 contributes to increased ER stress in APP/PS1 mice. a Western blot analysis of GRP78 and CHOP protein levels in hippocampal extracts of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b , c Data are presented as the mean ± SEM ( n = 3–5). The OD values of GRP78 ( b ) and CHOP ( c ) were normalized to that of β-actin. # p

    Journal: Journal of Neuroinflammation

    Article Title: Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling

    doi: 10.1186/s12974-018-1351-x

    Figure Lengend Snippet: KCa3.1 contributes to increased ER stress in APP/PS1 mice. a Western blot analysis of GRP78 and CHOP protein levels in hippocampal extracts of 15-month-old WT, KCa3.1 −/− , APP/PS1, and KCa3.1 −/− /APP/PS1 mice. b , c Data are presented as the mean ± SEM ( n = 3–5). The OD values of GRP78 ( b ) and CHOP ( c ) were normalized to that of β-actin. # p

    Article Snippet: The brain sections were then incubated with the following primary antibodies: rabbit anti-GFAP (1:1000; Dako, Glostrup, Denmark), mouse anti-GFAP (1:200; Merck Millipore, Burlington, MA, USA), rabbit anti-NeuN (1:100; Merck Millipore, Burlington, MA, USA), rabbit anti-Iba1 (1:500; Wako Pure Chemical Industries, Ltd., Osaka, Japan), mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel), rabbit anti-GRP78 (1:100; Cell Signaling Technology, Inc., Beverly, MA, USA), mouse anti-NG2 (Sigma-Aldrich Corporation, St. Louis, MO, USA), and rabbit anti-Orai1 (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Mouse Assay, Western Blot

    KCa3.1 involved in astrocytes SOCE and ER stress. a Primary cultured astrocytes were treated with 5 μM Aβ for 12 h with or without pretreatment of the KCa3.1 blocker TRAM-34 (1 μM). Fluorescence intensities of [Ca 2+ ] i are shown. Fluorescence intensity was measured in the presence of 1 μM Tg with or without 2 mM Ca 2+ . b Data are presented as the mean ± SEM ( n = 10). # p

    Journal: Journal of Neuroinflammation

    Article Title: Ca2+-dependent endoplasmic reticulum stress correlation with astrogliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling

    doi: 10.1186/s12974-018-1351-x

    Figure Lengend Snippet: KCa3.1 involved in astrocytes SOCE and ER stress. a Primary cultured astrocytes were treated with 5 μM Aβ for 12 h with or without pretreatment of the KCa3.1 blocker TRAM-34 (1 μM). Fluorescence intensities of [Ca 2+ ] i are shown. Fluorescence intensity was measured in the presence of 1 μM Tg with or without 2 mM Ca 2+ . b Data are presented as the mean ± SEM ( n = 10). # p

    Article Snippet: The brain sections were then incubated with the following primary antibodies: rabbit anti-GFAP (1:1000; Dako, Glostrup, Denmark), mouse anti-GFAP (1:200; Merck Millipore, Burlington, MA, USA), rabbit anti-NeuN (1:100; Merck Millipore, Burlington, MA, USA), rabbit anti-Iba1 (1:500; Wako Pure Chemical Industries, Ltd., Osaka, Japan), mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel), rabbit anti-GRP78 (1:100; Cell Signaling Technology, Inc., Beverly, MA, USA), mouse anti-NG2 (Sigma-Aldrich Corporation, St. Louis, MO, USA), and rabbit anti-Orai1 (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Cell Culture, Fluorescence

    Functional expression of SK4 channels in breast cancer cells. (A) Immunoblotting of SK4 and EMT-related proteins (E-cadherin and Vimentin) in breast cancer cell lines. (B) Comparison of SK4 mRNA expression in 4 breast cancer cell lines as determined by real-time PCR; n = 3. (C) Immunoblotting of ER protein in MDA-MB-468, MDA-MB-231 and T47D cells. (D-G) Immunostaining of SK4 (red) and nuclear marker DAPI (blue) in MDA-MB-231 (D), MDA-MB-468 (E), MCF-7 (F) and T47D (G) cells. Scale bars, 50 μm. (H, I) Whole-cell recording of MDA-MB-231 cells before (H) and after (I) 5-μM TRAM-34 treatment. (J, K) With (J) or without (K) 350 nM free Ca 2+ in the pipette solution, the voltage-current density curves show the currents changes before (a) and after (b) TRAM-34 treatment. The currents were evoked by step voltage ranging from -100 mV to +100 mV in steps of 10 mV every 100 ms. Dunnett’s Multiple Comparison Test was applied in comparison, ** p

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: Functional expression of SK4 channels in breast cancer cells. (A) Immunoblotting of SK4 and EMT-related proteins (E-cadherin and Vimentin) in breast cancer cell lines. (B) Comparison of SK4 mRNA expression in 4 breast cancer cell lines as determined by real-time PCR; n = 3. (C) Immunoblotting of ER protein in MDA-MB-468, MDA-MB-231 and T47D cells. (D-G) Immunostaining of SK4 (red) and nuclear marker DAPI (blue) in MDA-MB-231 (D), MDA-MB-468 (E), MCF-7 (F) and T47D (G) cells. Scale bars, 50 μm. (H, I) Whole-cell recording of MDA-MB-231 cells before (H) and after (I) 5-μM TRAM-34 treatment. (J, K) With (J) or without (K) 350 nM free Ca 2+ in the pipette solution, the voltage-current density curves show the currents changes before (a) and after (b) TRAM-34 treatment. The currents were evoked by step voltage ranging from -100 mV to +100 mV in steps of 10 mV every 100 ms. Dunnett’s Multiple Comparison Test was applied in comparison, ** p

    Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

    Techniques: Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Immunostaining, Marker, Transferring, Mass Spectrometry

    Blockage of SK4 channels inhibits MDA-MB-231 cell proliferation and colony formation ability, but not that of T47D cells. (A-D) Cell growth of MDA-MB-231 (A, B) and T47D cells (C, D) was analyzed using an MTT assay. The two cell lines were treated with 0–20 μM TRAM-34 (A, C) or clotrimazole (B, D) for 48 h, and the absorbance was measured; n = 5. (E, F) Images of the formed MDA-MB-231 colonies in the control group (CTL) and treatment groups (10 μM TRAM-34 and 20 μM TRAM-34); the bar represents separate counts of the colonies; n = 4. The data are presented as the mean ± SD, and Dunnett’s Multiple Comparison Test was applied in comparison. * p

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: Blockage of SK4 channels inhibits MDA-MB-231 cell proliferation and colony formation ability, but not that of T47D cells. (A-D) Cell growth of MDA-MB-231 (A, B) and T47D cells (C, D) was analyzed using an MTT assay. The two cell lines were treated with 0–20 μM TRAM-34 (A, C) or clotrimazole (B, D) for 48 h, and the absorbance was measured; n = 5. (E, F) Images of the formed MDA-MB-231 colonies in the control group (CTL) and treatment groups (10 μM TRAM-34 and 20 μM TRAM-34); the bar represents separate counts of the colonies; n = 4. The data are presented as the mean ± SD, and Dunnett’s Multiple Comparison Test was applied in comparison. * p

    Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

    Techniques: Multiple Displacement Amplification, MTT Assay, CTL Assay

    Blockage of SK4 channels promotes apoptosis in MDA-MB-231 cells but not T47D cells. MDA-MB-231 (A, B) and T47D (C, D) cells were treated with 20 μM TRAM-34 for 24 or 48 h, and cell apoptosis was analyzed by Annexin V-FITC/ PI-PE staining and flow cytometry. The bar of MDA-MB-231 indicates that the apoptosis rate of the TRAM-34-treated group increased apparently compared with that of the control (CTL). For T47D, the difference was not significant. The data are presented as the mean ± SD, and unpaired t test was applied in comparison. n = 3; * p

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: Blockage of SK4 channels promotes apoptosis in MDA-MB-231 cells but not T47D cells. MDA-MB-231 (A, B) and T47D (C, D) cells were treated with 20 μM TRAM-34 for 24 or 48 h, and cell apoptosis was analyzed by Annexin V-FITC/ PI-PE staining and flow cytometry. The bar of MDA-MB-231 indicates that the apoptosis rate of the TRAM-34-treated group increased apparently compared with that of the control (CTL). For T47D, the difference was not significant. The data are presented as the mean ± SD, and unpaired t test was applied in comparison. n = 3; * p

    Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

    Techniques: Multiple Displacement Amplification, Staining, Flow Cytometry, Cytometry, CTL Assay

    Down-regulation of SK4 channels inhibits the migration of MDA-MB-231 cells. A negative control siRNA (N.C.) and 3 SK4-specific siRNAs (Si-1, Si-2 and Si-3) were transfected into MDA-MB-231 cells, and 20 μM TRAM-34 was added to the TRAM-34-treated group to inhibit SK4 channels. (A, B) Knockdown of SK4 by siRNA was confirmed using immunoblotting and real-time PCR; n = 3. (C, D) The images and bar of the transwell migration assay indicate that the counts of migrated cells in SK4 siRNA (Si-SK4)- and TRAM-34-treated group were significantly less than those of the control (CTL). Scale bars, 50 μm; n = 4. (E, F) The images and bar of the wound-healing assay. The wound-healing rate represents the distance migrated by cells at certain time divided by the wound distance at 0 h. Scale bars, 100 μm; n = 3. The data are presented as the mean ± SD, Dunnett’s Multiple Comparison Test was applied in (B) and (D), and unpaired t test in (F). ** p

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: Down-regulation of SK4 channels inhibits the migration of MDA-MB-231 cells. A negative control siRNA (N.C.) and 3 SK4-specific siRNAs (Si-1, Si-2 and Si-3) were transfected into MDA-MB-231 cells, and 20 μM TRAM-34 was added to the TRAM-34-treated group to inhibit SK4 channels. (A, B) Knockdown of SK4 by siRNA was confirmed using immunoblotting and real-time PCR; n = 3. (C, D) The images and bar of the transwell migration assay indicate that the counts of migrated cells in SK4 siRNA (Si-SK4)- and TRAM-34-treated group were significantly less than those of the control (CTL). Scale bars, 50 μm; n = 4. (E, F) The images and bar of the wound-healing assay. The wound-healing rate represents the distance migrated by cells at certain time divided by the wound distance at 0 h. Scale bars, 100 μm; n = 3. The data are presented as the mean ± SD, Dunnett’s Multiple Comparison Test was applied in (B) and (D), and unpaired t test in (F). ** p

    Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

    Techniques: Migration, Multiple Displacement Amplification, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Transwell Migration Assay, CTL Assay, Wound Healing Assay

    The EGF/bFGF-induced EMT of MDA-MB-231 cells correlates with SK4 channels. (A) Phase contrast images of MDA-231 and T47D cells treated with (E+b) or without (CTL) EGF/bFGF for 1 day, 3 days and 5 days. Scale bars, 100 μm. (B, C) The EGF/bFGF-induced EMT of MDA-231 cells was confirmed using immunoblotting and real-time PCR of EMT markers (Vimentin, Snail1 and Slug), and the SK4 mRNA level increased after EMT. (D) Immunoblotting of EMT-related proteins (Vimentin and Snail1) was performed 72 h after MDA-231 cells were transfected with negative control siRNA (N.C.) or SK4-specific siRNA (Si-SK4); cells that did not undergo transfection served as a control (CTL). The data are presented as the mean ± SD, and paired t test was applied in comparison. n = 3; * p

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: The EGF/bFGF-induced EMT of MDA-MB-231 cells correlates with SK4 channels. (A) Phase contrast images of MDA-231 and T47D cells treated with (E+b) or without (CTL) EGF/bFGF for 1 day, 3 days and 5 days. Scale bars, 100 μm. (B, C) The EGF/bFGF-induced EMT of MDA-231 cells was confirmed using immunoblotting and real-time PCR of EMT markers (Vimentin, Snail1 and Slug), and the SK4 mRNA level increased after EMT. (D) Immunoblotting of EMT-related proteins (Vimentin and Snail1) was performed 72 h after MDA-231 cells were transfected with negative control siRNA (N.C.) or SK4-specific siRNA (Si-SK4); cells that did not undergo transfection served as a control (CTL). The data are presented as the mean ± SD, and paired t test was applied in comparison. n = 3; * p

    Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

    Techniques: Multiple Displacement Amplification, CTL Assay, Real-time Polymerase Chain Reaction, Transfection, Negative Control

    SK4 proteins expressed in breast cancer tissue. (A-D) SK4 IHC in fours subtypes of breast cancer tissues including Luminal A (A), Luminal B (B), HER2 (C), and TNBC (D). Scale bars, 50 μm. (E) Immunoblotting of SK4 and E-cadherin in breast cancer tissues (BC1 and BC2) and non-tumor breast tissues (Non-Tumor1 and Non-Tumor2).

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: SK4 proteins expressed in breast cancer tissue. (A-D) SK4 IHC in fours subtypes of breast cancer tissues including Luminal A (A), Luminal B (B), HER2 (C), and TNBC (D). Scale bars, 50 μm. (E) Immunoblotting of SK4 and E-cadherin in breast cancer tissues (BC1 and BC2) and non-tumor breast tissues (Non-Tumor1 and Non-Tumor2).

    Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

    Techniques: Immunohistochemistry