orai1  (Alomone Labs)


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    Alomone Labs orai1
    Effect of <t>Orai1</t> small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Orai1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/orai1/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    orai1 - by Bioz Stars, 2022-01
    92/100 stars

    Images

    1) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    2) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    3) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p

    Techniques Used: Expressing

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

    4) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

    5) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    6) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    7) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    8) Product Images from "Chronic Hypoxia Increases TRPC6 Expression and Basal Intracellular Ca2+ Concentration in Rat Distal Pulmonary Venous Smooth Muscle"

    Article Title: Chronic Hypoxia Increases TRPC6 Expression and Basal Intracellular Ca2+ Concentration in Rat Distal Pulmonary Venous Smooth Muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112007

    Effects of siOrai1 transfection on basal [Ca 2+ ] i and SOCE in rat PVSMCs. A, B : Western blot showing expression of Orai1 and α-actin protein in rat PVSMC treated with siNT, siOrai1 and transfection vehicle alone (control). E, F : The changes of SOCE in siOrai1, NTsiRNA transfected PVSMCs and transfection vehicle alone treated control. *P
    Figure Legend Snippet: Effects of siOrai1 transfection on basal [Ca 2+ ] i and SOCE in rat PVSMCs. A, B : Western blot showing expression of Orai1 and α-actin protein in rat PVSMC treated with siNT, siOrai1 and transfection vehicle alone (control). E, F : The changes of SOCE in siOrai1, NTsiRNA transfected PVSMCs and transfection vehicle alone treated control. *P

    Techniques Used: Transfection, Western Blot, Expressing

    9) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    10) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    11) Product Images from "Store-Operated Calcium Channels Are Involved in Spontaneous Slow Calcium Oscillations in Striatal Neurons"

    Article Title: Store-Operated Calcium Channels Are Involved in Spontaneous Slow Calcium Oscillations in Striatal Neurons

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2019.00547

    Each of the molecular components of SOCE expressed at different levels in the striatal GFP-positive cells. Confocal microscopy images of the sections through the dorsal striatum obtained from GAD67-GFP mice, which were stained for Orai1, Orai 2, Orai3, STIM1, or STIM2 (red), along with GFP (green). Blue staining represents DAPI. Scale bar: 50 μm.
    Figure Legend Snippet: Each of the molecular components of SOCE expressed at different levels in the striatal GFP-positive cells. Confocal microscopy images of the sections through the dorsal striatum obtained from GAD67-GFP mice, which were stained for Orai1, Orai 2, Orai3, STIM1, or STIM2 (red), along with GFP (green). Blue staining represents DAPI. Scale bar: 50 μm.

    Techniques Used: Confocal Microscopy, Mouse Assay, Staining

    12) Product Images from "CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission"

    Article Title: CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission

    Journal: Science signaling

    doi: 10.1126/scisignal.aaw5450

    Orai1 channels generate GPCR-mediated Ca 2 +fluctuations in astrocyte processes in situ. ( A ) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB 1 D promoter. After 2 to 3 weeks, to allow for expression, Ca 2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. ( B and C ) Images of GCaMP6f-expressing WT ( Orai1 fl/fl ) (B) or Orai1 KO ( Orai1 fl/fl GFAP-Cre ) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca 2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein-coupled PARs on astrocytes and evoke Ca 2+ signaling. Movies of the Ca 2+ . ( D and E ) Summary of the Ca 2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT ( Orai1 fl/fl , black bars) and Orai1 KO ( Orail fl/fl GFAP-Cre , orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. ( F and G ) Cumulative probability plots of the amplitudes of each Ca 2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. ( H and I ) Comparison of WT and Orai1 KO Ca 2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca 2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Orai1 channels generate GPCR-mediated Ca 2 +fluctuations in astrocyte processes in situ. ( A ) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB 1 D promoter. After 2 to 3 weeks, to allow for expression, Ca 2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. ( B and C ) Images of GCaMP6f-expressing WT ( Orai1 fl/fl ) (B) or Orai1 KO ( Orai1 fl/fl GFAP-Cre ) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca 2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein-coupled PARs on astrocytes and evoke Ca 2+ signaling. Movies of the Ca 2+ . ( D and E ) Summary of the Ca 2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT ( Orai1 fl/fl , black bars) and Orai1 KO ( Orail fl/fl GFAP-Cre , orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. ( F and G ) Cumulative probability plots of the amplitudes of each Ca 2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. ( H and I ) Comparison of WT and Orai1 KO Ca 2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca 2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. * P

    Techniques Used: In Situ, Expressing, Mouse Assay

    Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes. ( A ) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca 2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. ( B ) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes. ( A ) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca 2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. ( B ) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. * P

    Techniques Used: Cell Culture, Luciferase, Luminescence Assay

    Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes. ( A ) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca 2+ -free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca 2+ elicited SOCE that was significantly decreased in Orail KO ( Orai1 fl/fl nestln-Cre and Orai1 fl/fl GFAP-Cre ) cells, as measured by the rate of Ca 2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. ( B ) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca 2+ -free solution and subsequent sustained SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca 2+ -free solution followed by SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. *** P
    Figure Legend Snippet: Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes. ( A ) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca 2+ -free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca 2+ elicited SOCE that was significantly decreased in Orail KO ( Orai1 fl/fl nestln-Cre and Orai1 fl/fl GFAP-Cre ) cells, as measured by the rate of Ca 2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. ( B ) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca 2+ -free solution and subsequent sustained SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca 2+ -free solution followed by SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. *** P

    Techniques Used: Cell Culture

    Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1. ( A ) Depletion of ER Ca 2+ stores with TG (1 μM) in a Ca 2+ -free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca 2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl 3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca 2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca 2+ influx rates were calculated by measuring the initial slope of Ca 2 + entry over 18 s after readdition of 2 mM Ca 2+ solution (as shown by the dotted line for the control condition). ( B ) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca 2+ influx rates were attenuated after readdition of external Ca 2+ . The right graph shows the summary of the rate of Ca 2+ influx in WT ( Orai1 fl/+ and Orai1 fl / fl ), Orai1 HET ( Orai1 fl/+ nest,n-Cre and Orai1 fl /− ), and Orai1 KO ( Orai1 fl/fl nestin-Cre and Orai1 fl/−nestin-Cre ) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. ( C ) SOCE was abolished in cultured astrocytes from STIM1 KO mice ( STIM1 fl/fl nestin-Cre ). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. ( D ) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice ( Orai1 fl/fl GFAP-Cre ). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. ** P
    Figure Legend Snippet: Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1. ( A ) Depletion of ER Ca 2+ stores with TG (1 μM) in a Ca 2+ -free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca 2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl 3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca 2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca 2+ influx rates were calculated by measuring the initial slope of Ca 2 + entry over 18 s after readdition of 2 mM Ca 2+ solution (as shown by the dotted line for the control condition). ( B ) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca 2+ influx rates were attenuated after readdition of external Ca 2+ . The right graph shows the summary of the rate of Ca 2+ influx in WT ( Orai1 fl/+ and Orai1 fl / fl ), Orai1 HET ( Orai1 fl/+ nest,n-Cre and Orai1 fl /− ), and Orai1 KO ( Orai1 fl/fl nestin-Cre and Orai1 fl/−nestin-Cre ) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. ( C ) SOCE was abolished in cultured astrocytes from STIM1 KO mice ( STIM1 fl/fl nestin-Cre ). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. ( D ) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice ( Orai1 fl/fl GFAP-Cre ). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. ** P

    Techniques Used: Cell Culture, Mouse Assay

    Orai1 channels stimulate vesicular exocytosis after store depletion. ( A ) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 mm. ( B ) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT ( Orai1 fl/+ ) astrocyte. Scale bar, 20 μm. ( C ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. ( D ) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orail KO ( Orai1 fl/fl GFAP-Cre ) astrocyte. Scale bar, 20 μm. ( E ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. ( F ) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. ( G ) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orail KO cells, by preincubation with BAPTA-AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orail KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Orai1 channels stimulate vesicular exocytosis after store depletion. ( A ) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 mm. ( B ) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT ( Orai1 fl/+ ) astrocyte. Scale bar, 20 μm. ( C ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. ( D ) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orail KO ( Orai1 fl/fl GFAP-Cre ) astrocyte. Scale bar, 20 μm. ( E ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. ( F ) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. ( G ) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orail KO cells, by preincubation with BAPTA-AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orail KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. * P

    Techniques Used: Fluorescence

    Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes. ( A ) Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. ( B ) Left: Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. ( C and D ) Location of spH events mapped onto the footprint of an Orai1 KO ( Orai1 fl/fl GFAP-Cre ) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). ( E ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by UTP. ( F ) The average rate of UTP-evoked exocytosis per 1000 μm 2 was significantly suppressed in Orai1 KO cells, in Ca 2+ -free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca 2+ -free, n = 10 cells; TeTx, n = 7 cells. ( G ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm 2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca 2+ -free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes. ( A ) Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. ( B ) Left: Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. ( C and D ) Location of spH events mapped onto the footprint of an Orai1 KO ( Orai1 fl/fl GFAP-Cre ) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). ( E ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by UTP. ( F ) The average rate of UTP-evoked exocytosis per 1000 μm 2 was significantly suppressed in Orai1 KO cells, in Ca 2+ -free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca 2+ -free, n = 10 cells; TeTx, n = 7 cells. ( G ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm 2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca 2+ -free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. * P

    Techniques Used: Expressing

    Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells. ( A ) Administration of thrombin evokes a burst of spontaneous IPSCs on Orail (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. ( B ) sIPSC traces from the experiment in (A) shown on an expanded timescale. ( C ) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (* P = 0.02 by paired t test, n = 8 cells). ( D ) Amplitude distribution of the sIPSC events in WT slices. ( E ) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX ( n = 8 cells). ( F ) The thrombin- induced sIPSC response in CA1 neurons is abolished in Orai1 fl/fl GFAP-Cre slices. ( G ) sIPSC traces from the experiment in (F) shown on an expanded timescale. ( H ) Summary of sIPSC frequency and amplitude in Orai1 fl/fl GFAP-Cre slices before and after application of thrombin ( n = 6 cells). ( I ) Amplitude distribution of the sIPSC events in Orai1 KO slices. ( J ) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices ( n = 4 cells). Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells. ( A ) Administration of thrombin evokes a burst of spontaneous IPSCs on Orail (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. ( B ) sIPSC traces from the experiment in (A) shown on an expanded timescale. ( C ) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (* P = 0.02 by paired t test, n = 8 cells). ( D ) Amplitude distribution of the sIPSC events in WT slices. ( E ) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX ( n = 8 cells). ( F ) The thrombin- induced sIPSC response in CA1 neurons is abolished in Orai1 fl/fl GFAP-Cre slices. ( G ) sIPSC traces from the experiment in (F) shown on an expanded timescale. ( H ) Summary of sIPSC frequency and amplitude in Orai1 fl/fl GFAP-Cre slices before and after application of thrombin ( n = 6 cells). ( I ) Amplitude distribution of the sIPSC events in Orai1 KO slices. ( J ) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices ( n = 4 cells). Bar graphs show means ± SEM. * P

    Techniques Used: Patch Clamp, Isolation

    13) Product Images from "ASIC1-mediated calcium entry stimulates NFATc3 nuclear translocation via PICK1 coupling in pulmonary arterial smooth muscle cells"

    Article Title: ASIC1-mediated calcium entry stimulates NFATc3 nuclear translocation via PICK1 coupling in pulmonary arterial smooth muscle cells

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00040.2016

    Knockout of ASIC1 does not alter NFAT, Orai1, or TRPC1 mRNA and protein levels in PASMC. Fold change (from calibrator) in NFATc2 ( A ), NFATc3 ( B ), Orai1 ( C ), and TRPC1 ( D ) mRNA expression in PASMC from ASIC1 +/+ and ASIC1 −/− mice. β-Actin
    Figure Legend Snippet: Knockout of ASIC1 does not alter NFAT, Orai1, or TRPC1 mRNA and protein levels in PASMC. Fold change (from calibrator) in NFATc2 ( A ), NFATc3 ( B ), Orai1 ( C ), and TRPC1 ( D ) mRNA expression in PASMC from ASIC1 +/+ and ASIC1 −/− mice. β-Actin

    Techniques Used: Knock-Out, Expressing, Mouse Assay

    14) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

    15) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    16) Product Images from "The plasma membrane channel ORAI1 mediates detrimental calcium influx caused by endogenous oxidative stress"

    Article Title: The plasma membrane channel ORAI1 mediates detrimental calcium influx caused by endogenous oxidative stress

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.216

    The SOCE Ca 2+ influx channel ORAI1 is downregulated in glutamate-resistant HT22R cells. Expression analysis of key SOCE players and TRPM7 in HT22S and R cells. (a, b and d) Immunoblots were probed with antibodies against STIM1 (a) or 2 (b) and ORAI1 (d) ; GAPDH served as loading control and was simultaneously recorded with the protein of interest on the same membrane with the Licor infrared imaging system. Quantification of immunoblots was done with ImageJ software. Intensity values were normalized to HT22S samples and plotted in bar graphs as expression in percentage. (c) RT-PCR analysis of TRPM7 expression level in HT22S and R cells. The TRPM7 signal was normalized to the endogenous control hprt and plotted as mean expression level±S.E.M. in percentage. All graphs show the mean of at least three independently performed experiments. * P
    Figure Legend Snippet: The SOCE Ca 2+ influx channel ORAI1 is downregulated in glutamate-resistant HT22R cells. Expression analysis of key SOCE players and TRPM7 in HT22S and R cells. (a, b and d) Immunoblots were probed with antibodies against STIM1 (a) or 2 (b) and ORAI1 (d) ; GAPDH served as loading control and was simultaneously recorded with the protein of interest on the same membrane with the Licor infrared imaging system. Quantification of immunoblots was done with ImageJ software. Intensity values were normalized to HT22S samples and plotted in bar graphs as expression in percentage. (c) RT-PCR analysis of TRPM7 expression level in HT22S and R cells. The TRPM7 signal was normalized to the endogenous control hprt and plotted as mean expression level±S.E.M. in percentage. All graphs show the mean of at least three independently performed experiments. * P

    Techniques Used: Expressing, Western Blot, Imaging, Software, Reverse Transcription Polymerase Chain Reaction

    Knockdown of ORAI1 inhibits cytosolic calcium elevation during oxytosis. HT22S cells were transfected with two different siRNAs against ORAI1 or non-targeting siRNA as control together with a plasmid encoding the GCaMP5-IRES-RFP Ca 2+ reporter gene and subjected to long-term live-cell imaging experiments to show the detrimental Ca 2+ influx in the late phase of oxidative glutamate toxicity. (a) Typical GCaMP pictures of a control siRNA transfected cell under glutamate exposure. The cytosolic Ca 2+ concentration stays low for several hours, but eventually rises quickly, leading to cell lysis assessed by quenching of the fluorescence signal. The lower panel shows sample traces from glutamate-treated cells transfected with control siRNA, which illustrate the asynchronous appearance of Ca 2+ -spikes. For reasons of clarity, only Ca 2+ -peak positive cells from one well are shown. (b) Analysis of long-term live-cell imaging of GCaMP-IRES-RFP and siRNA-transfected cells under glutamate exposure in comparison to vehicle treatment. For each picture, a GCaMP5/RFP ratio was calculated. To identify peaking cells, the maximal GCaMP/RFP ratio was divided by the average GCaMP/RFP ratio calculated over the whole measurement period. Each cell that reached a value above the average value of control siRNA transfected cells was counted as Ca 2+ -peak positive. The amount of Ca 2+ -peaking cells was normalized to vehicle control and plotted as a bar graph. The bars represent mean±S.E.M. of 15 replicate wells measured in three independent experiments. (c) Analysis of the increase in the GCaMP/RFP ratio in pCPT-cGMP treated HT22 cells transfected with ORAI1 or control siRNA. The slope was normalized to vehicle and plotted as mean±S.E.M. in bar graphs. Two independent experiments were performed containing > 150 cells for each condition. N.S., not significant, * P
    Figure Legend Snippet: Knockdown of ORAI1 inhibits cytosolic calcium elevation during oxytosis. HT22S cells were transfected with two different siRNAs against ORAI1 or non-targeting siRNA as control together with a plasmid encoding the GCaMP5-IRES-RFP Ca 2+ reporter gene and subjected to long-term live-cell imaging experiments to show the detrimental Ca 2+ influx in the late phase of oxidative glutamate toxicity. (a) Typical GCaMP pictures of a control siRNA transfected cell under glutamate exposure. The cytosolic Ca 2+ concentration stays low for several hours, but eventually rises quickly, leading to cell lysis assessed by quenching of the fluorescence signal. The lower panel shows sample traces from glutamate-treated cells transfected with control siRNA, which illustrate the asynchronous appearance of Ca 2+ -spikes. For reasons of clarity, only Ca 2+ -peak positive cells from one well are shown. (b) Analysis of long-term live-cell imaging of GCaMP-IRES-RFP and siRNA-transfected cells under glutamate exposure in comparison to vehicle treatment. For each picture, a GCaMP5/RFP ratio was calculated. To identify peaking cells, the maximal GCaMP/RFP ratio was divided by the average GCaMP/RFP ratio calculated over the whole measurement period. Each cell that reached a value above the average value of control siRNA transfected cells was counted as Ca 2+ -peak positive. The amount of Ca 2+ -peaking cells was normalized to vehicle control and plotted as a bar graph. The bars represent mean±S.E.M. of 15 replicate wells measured in three independent experiments. (c) Analysis of the increase in the GCaMP/RFP ratio in pCPT-cGMP treated HT22 cells transfected with ORAI1 or control siRNA. The slope was normalized to vehicle and plotted as mean±S.E.M. in bar graphs. Two independent experiments were performed containing > 150 cells for each condition. N.S., not significant, * P

    Techniques Used: Transfection, Plasmid Preparation, Live Cell Imaging, Concentration Assay, Lysis, Fluorescence

    Knockdown of ORAI1 protects from GSH depletion. HT22S cells were transfected with two independent siRNAs against STIM1, STIM2, ORAI1 and TRPM7 or with non-targeting control siRNA and 48 h later subjected to cell survival experiments. (a, d, f and h) RT-PCR analyses of successful knockdown of STIM1 (a) , STIM2 (d) , ORAI1 (h) and TRPM7 (f) . Signals of genes of interest were normalized to hprt and plotted as expression in percentage relative to control-siRNA transfected samples. (b and i) Verification of successful knockdown by immunoblot. The membranes were simultaneously probed with antibody against either STIM1 (b) or ORAI1 (i) and GAPDH respectively actin as loading control and analyzed with the Licor infrared imaging system. (c, e, g and j) Survival experiments of siRNA-transfected cells. The indicated amounts of glutamate or BSO were added 48 h after transfection and viability was quantified with the CTB reagent 16 h after glutamate or BSO addition. The graphs show the mean fluorescence±S.E.M. of 15 replicates obtained in three independent experiments plotted against glutamate or BSO concentration
    Figure Legend Snippet: Knockdown of ORAI1 protects from GSH depletion. HT22S cells were transfected with two independent siRNAs against STIM1, STIM2, ORAI1 and TRPM7 or with non-targeting control siRNA and 48 h later subjected to cell survival experiments. (a, d, f and h) RT-PCR analyses of successful knockdown of STIM1 (a) , STIM2 (d) , ORAI1 (h) and TRPM7 (f) . Signals of genes of interest were normalized to hprt and plotted as expression in percentage relative to control-siRNA transfected samples. (b and i) Verification of successful knockdown by immunoblot. The membranes were simultaneously probed with antibody against either STIM1 (b) or ORAI1 (i) and GAPDH respectively actin as loading control and analyzed with the Licor infrared imaging system. (c, e, g and j) Survival experiments of siRNA-transfected cells. The indicated amounts of glutamate or BSO were added 48 h after transfection and viability was quantified with the CTB reagent 16 h after glutamate or BSO addition. The graphs show the mean fluorescence±S.E.M. of 15 replicates obtained in three independent experiments plotted against glutamate or BSO concentration

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Imaging, CtB Assay, Fluorescence, Concentration Assay

    17) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    18) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    19) Product Images from "CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission"

    Article Title: CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission

    Journal: Science signaling

    doi: 10.1126/scisignal.aaw5450

    Orai1 channels generate GPCR-mediated Ca 2 +fluctuations in astrocyte processes in situ. ( A ) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB 1 D promoter. After 2 to 3 weeks, to allow for expression, Ca 2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. ( B and C ) Images of GCaMP6f-expressing WT ( Orai1 fl/fl ) (B) or Orai1 KO ( Orai1 fl/fl GFAP-Cre ) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca 2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein-coupled PARs on astrocytes and evoke Ca 2+ signaling. Movies of the Ca 2+ . ( D and E ) Summary of the Ca 2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT ( Orai1 fl/fl , black bars) and Orai1 KO ( Orail fl/fl GFAP-Cre , orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. ( F and G ) Cumulative probability plots of the amplitudes of each Ca 2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. ( H and I ) Comparison of WT and Orai1 KO Ca 2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca 2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Orai1 channels generate GPCR-mediated Ca 2 +fluctuations in astrocyte processes in situ. ( A ) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB 1 D promoter. After 2 to 3 weeks, to allow for expression, Ca 2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. ( B and C ) Images of GCaMP6f-expressing WT ( Orai1 fl/fl ) (B) or Orai1 KO ( Orai1 fl/fl GFAP-Cre ) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca 2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein-coupled PARs on astrocytes and evoke Ca 2+ signaling. Movies of the Ca 2+ . ( D and E ) Summary of the Ca 2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT ( Orai1 fl/fl , black bars) and Orai1 KO ( Orail fl/fl GFAP-Cre , orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. ( F and G ) Cumulative probability plots of the amplitudes of each Ca 2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. ( H and I ) Comparison of WT and Orai1 KO Ca 2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca 2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. * P

    Techniques Used: In Situ, Expressing, Mouse Assay

    Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes. ( A ) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca 2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. ( B ) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes. ( A ) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca 2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. ( B ) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. * P

    Techniques Used: Cell Culture, Luciferase, Luminescence Assay

    Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes. ( A ) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca 2+ -free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca 2+ elicited SOCE that was significantly decreased in Orail KO ( Orai1 fl/fl nestln-Cre and Orai1 fl/fl GFAP-Cre ) cells, as measured by the rate of Ca 2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. ( B ) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca 2+ -free solution and subsequent sustained SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca 2+ -free solution followed by SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. *** P
    Figure Legend Snippet: Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes. ( A ) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca 2+ -free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca 2+ elicited SOCE that was significantly decreased in Orail KO ( Orai1 fl/fl nestln-Cre and Orai1 fl/fl GFAP-Cre ) cells, as measured by the rate of Ca 2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. ( B ) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca 2+ -free solution and subsequent sustained SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca 2+ -free solution followed by SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. *** P

    Techniques Used: Cell Culture

    Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1. ( A ) Depletion of ER Ca 2+ stores with TG (1 μM) in a Ca 2+ -free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca 2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl 3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca 2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca 2+ influx rates were calculated by measuring the initial slope of Ca 2 + entry over 18 s after readdition of 2 mM Ca 2+ solution (as shown by the dotted line for the control condition). ( B ) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca 2+ influx rates were attenuated after readdition of external Ca 2+ . The right graph shows the summary of the rate of Ca 2+ influx in WT ( Orai1 fl/+ and Orai1 fl / fl ), Orai1 HET ( Orai1 fl/+ nest,n-Cre and Orai1 fl /− ), and Orai1 KO ( Orai1 fl/fl nestin-Cre and Orai1 fl/−nestin-Cre ) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. ( C ) SOCE was abolished in cultured astrocytes from STIM1 KO mice ( STIM1 fl/fl nestin-Cre ). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. ( D ) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice ( Orai1 fl/fl GFAP-Cre ). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. ** P
    Figure Legend Snippet: Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1. ( A ) Depletion of ER Ca 2+ stores with TG (1 μM) in a Ca 2+ -free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca 2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl 3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca 2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca 2+ influx rates were calculated by measuring the initial slope of Ca 2 + entry over 18 s after readdition of 2 mM Ca 2+ solution (as shown by the dotted line for the control condition). ( B ) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca 2+ influx rates were attenuated after readdition of external Ca 2+ . The right graph shows the summary of the rate of Ca 2+ influx in WT ( Orai1 fl/+ and Orai1 fl / fl ), Orai1 HET ( Orai1 fl/+ nest,n-Cre and Orai1 fl /− ), and Orai1 KO ( Orai1 fl/fl nestin-Cre and Orai1 fl/−nestin-Cre ) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. ( C ) SOCE was abolished in cultured astrocytes from STIM1 KO mice ( STIM1 fl/fl nestin-Cre ). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. ( D ) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice ( Orai1 fl/fl GFAP-Cre ). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. ** P

    Techniques Used: Cell Culture, Mouse Assay

    Orai1 channels stimulate vesicular exocytosis after store depletion. ( A ) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 mm. ( B ) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT ( Orai1 fl/+ ) astrocyte. Scale bar, 20 μm. ( C ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. ( D ) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orail KO ( Orai1 fl/fl GFAP-Cre ) astrocyte. Scale bar, 20 μm. ( E ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. ( F ) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. ( G ) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orail KO cells, by preincubation with BAPTA-AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orail KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Orai1 channels stimulate vesicular exocytosis after store depletion. ( A ) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 mm. ( B ) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT ( Orai1 fl/+ ) astrocyte. Scale bar, 20 μm. ( C ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. ( D ) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orail KO ( Orai1 fl/fl GFAP-Cre ) astrocyte. Scale bar, 20 μm. ( E ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. ( F ) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. ( G ) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orail KO cells, by preincubation with BAPTA-AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orail KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. * P

    Techniques Used: Fluorescence

    Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes. ( A ) Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. ( B ) Left: Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. ( C and D ) Location of spH events mapped onto the footprint of an Orai1 KO ( Orai1 fl/fl GFAP-Cre ) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). ( E ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by UTP. ( F ) The average rate of UTP-evoked exocytosis per 1000 μm 2 was significantly suppressed in Orai1 KO cells, in Ca 2+ -free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca 2+ -free, n = 10 cells; TeTx, n = 7 cells. ( G ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm 2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca 2+ -free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes. ( A ) Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. ( B ) Left: Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. ( C and D ) Location of spH events mapped onto the footprint of an Orai1 KO ( Orai1 fl/fl GFAP-Cre ) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). ( E ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by UTP. ( F ) The average rate of UTP-evoked exocytosis per 1000 μm 2 was significantly suppressed in Orai1 KO cells, in Ca 2+ -free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca 2+ -free, n = 10 cells; TeTx, n = 7 cells. ( G ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm 2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca 2+ -free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. * P

    Techniques Used: Expressing

    Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells. ( A ) Administration of thrombin evokes a burst of spontaneous IPSCs on Orail (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. ( B ) sIPSC traces from the experiment in (A) shown on an expanded timescale. ( C ) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (* P = 0.02 by paired t test, n = 8 cells). ( D ) Amplitude distribution of the sIPSC events in WT slices. ( E ) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX ( n = 8 cells). ( F ) The thrombin- induced sIPSC response in CA1 neurons is abolished in Orai1 fl/fl GFAP-Cre slices. ( G ) sIPSC traces from the experiment in (F) shown on an expanded timescale. ( H ) Summary of sIPSC frequency and amplitude in Orai1 fl/fl GFAP-Cre slices before and after application of thrombin ( n = 6 cells). ( I ) Amplitude distribution of the sIPSC events in Orai1 KO slices. ( J ) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices ( n = 4 cells). Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells. ( A ) Administration of thrombin evokes a burst of spontaneous IPSCs on Orail (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. ( B ) sIPSC traces from the experiment in (A) shown on an expanded timescale. ( C ) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (* P = 0.02 by paired t test, n = 8 cells). ( D ) Amplitude distribution of the sIPSC events in WT slices. ( E ) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX ( n = 8 cells). ( F ) The thrombin- induced sIPSC response in CA1 neurons is abolished in Orai1 fl/fl GFAP-Cre slices. ( G ) sIPSC traces from the experiment in (F) shown on an expanded timescale. ( H ) Summary of sIPSC frequency and amplitude in Orai1 fl/fl GFAP-Cre slices before and after application of thrombin ( n = 6 cells). ( I ) Amplitude distribution of the sIPSC events in Orai1 KO slices. ( J ) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices ( n = 4 cells). Bar graphs show means ± SEM. * P

    Techniques Used: Patch Clamp, Isolation

    20) Product Images from "Inhibition of Orai1‐mediated Ca2+ entry enhances chemosensitivity of HepG2 hepatocarcinoma cells to 5‐fluorouracil"

    Article Title: Inhibition of Orai1‐mediated Ca2+ entry enhances chemosensitivity of HepG2 hepatocarcinoma cells to 5‐fluorouracil

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13029

    Orai1 inhibits 5‐ FU ‐induced autophagy through AKT / mTOR signalling pathway. ( A ) HepG2 cells were treated with Orai1 si RNA (50 nM) for 48 hrs or treated with SKF 96365 (20 μM) for 3 hrs prior to 5‐ FU (80 μM) incubation for another 48 hrs. The phosphorylation of AKT , mTOR and p70S6K were determined by Western blotting. ( B ) Bar diagram represents the densitometric analysis of the phosphorylation level of above proteins. ** P
    Figure Legend Snippet: Orai1 inhibits 5‐ FU ‐induced autophagy through AKT / mTOR signalling pathway. ( A ) HepG2 cells were treated with Orai1 si RNA (50 nM) for 48 hrs or treated with SKF 96365 (20 μM) for 3 hrs prior to 5‐ FU (80 μM) incubation for another 48 hrs. The phosphorylation of AKT , mTOR and p70S6K were determined by Western blotting. ( B ) Bar diagram represents the densitometric analysis of the phosphorylation level of above proteins. ** P

    Techniques Used: Incubation, Western Blot

    Restoration of Orai1 attenuates 5‐ FU ‐induced autophagic cell death. ( A ) Cells were transfected with Orai1 plasmid for 48 hrs prior to 5‐ FU (80 μM) incubation for another 48 hrs. Ca 2+ imaging experiment was performed as mentioned in methods section. ( B ) Quantification of fluorescence ratio (340/380). ** P
    Figure Legend Snippet: Restoration of Orai1 attenuates 5‐ FU ‐induced autophagic cell death. ( A ) Cells were transfected with Orai1 plasmid for 48 hrs prior to 5‐ FU (80 μM) incubation for another 48 hrs. Ca 2+ imaging experiment was performed as mentioned in methods section. ( B ) Quantification of fluorescence ratio (340/380). ** P

    Techniques Used: Transfection, Plasmid Preparation, Incubation, Imaging, Fluorescence

    Inhibition of Orai1 or SOCE enhances 5‐ FU ‐induced autophagy and cell death. ( A ) Cells were treated with Orai1 si RNA (50 nM ) for 48 hrs or treated with SKF 96365 (20 μM) for 3 hrs prior to 5‐ FU (80 μM) incubation. At the end of the experiment, cells were loaded with Fura2/ AM probe and subjected to Ca 2+ imaging experiment. ( B ) Quantification of fluorescence ratio (340/380). ** P
    Figure Legend Snippet: Inhibition of Orai1 or SOCE enhances 5‐ FU ‐induced autophagy and cell death. ( A ) Cells were treated with Orai1 si RNA (50 nM ) for 48 hrs or treated with SKF 96365 (20 μM) for 3 hrs prior to 5‐ FU (80 μM) incubation. At the end of the experiment, cells were loaded with Fura2/ AM probe and subjected to Ca 2+ imaging experiment. ( B ) Quantification of fluorescence ratio (340/380). ** P

    Techniques Used: Inhibition, Incubation, Imaging, Fluorescence

    5‐ FU inhibits SOCE through reducing Orai1 expression. ( A ) Cells were treated with various concentrations of 5‐ FU for 48 hrs. Ca 2+ images showing the thapsigargin (Tg, 1 μM)‐induced Ca 2+ entry after addition of 1 mM Ca 2+ in Ca 2+ ‐free medium. ( B ) Bar diagram showing the fluorescence ratio (340/380) as mentioned in methods section. * P
    Figure Legend Snippet: 5‐ FU inhibits SOCE through reducing Orai1 expression. ( A ) Cells were treated with various concentrations of 5‐ FU for 48 hrs. Ca 2+ images showing the thapsigargin (Tg, 1 μM)‐induced Ca 2+ entry after addition of 1 mM Ca 2+ in Ca 2+ ‐free medium. ( B ) Bar diagram showing the fluorescence ratio (340/380) as mentioned in methods section. * P

    Techniques Used: Expressing, Fluorescence

    Orai1 is overexpressed in liver cancer tissues and involved in SOCE in HepG2 cells. ( A ) Histopathological changes in liver cancer and adjacent normal tissues from same hepatocarcinoma patients were examined by haematoxylin and eosin staining, and Orai1 expression was determined by immunohistochemistry. ( B ) Representative Western blotting analysis of Orai1 expression in liver cancer samples. ( C ) Densitometric analysis of Orai1 protein expression. ( D ) mRNA expression of Orai1 was analysed by quantitative real‐time PCR . ** P
    Figure Legend Snippet: Orai1 is overexpressed in liver cancer tissues and involved in SOCE in HepG2 cells. ( A ) Histopathological changes in liver cancer and adjacent normal tissues from same hepatocarcinoma patients were examined by haematoxylin and eosin staining, and Orai1 expression was determined by immunohistochemistry. ( B ) Representative Western blotting analysis of Orai1 expression in liver cancer samples. ( C ) Densitometric analysis of Orai1 protein expression. ( D ) mRNA expression of Orai1 was analysed by quantitative real‐time PCR . ** P

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

    21) Product Images from "CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission"

    Article Title: CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission

    Journal: Science signaling

    doi: 10.1126/scisignal.aaw5450

    Orai1 channels generate GPCR-mediated Ca 2 +fluctuations in astrocyte processes in situ. ( A ) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB 1 D promoter. After 2 to 3 weeks, to allow for expression, Ca 2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. ( B and C ) Images of GCaMP6f-expressing WT ( Orai1 fl/fl ) (B) or Orai1 KO ( Orai1 fl/fl GFAP-Cre ) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca 2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein-coupled PARs on astrocytes and evoke Ca 2+ signaling. Movies of the Ca 2+ . ( D and E ) Summary of the Ca 2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT ( Orai1 fl/fl , black bars) and Orai1 KO ( Orail fl/fl GFAP-Cre , orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. ( F and G ) Cumulative probability plots of the amplitudes of each Ca 2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. ( H and I ) Comparison of WT and Orai1 KO Ca 2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca 2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Orai1 channels generate GPCR-mediated Ca 2 +fluctuations in astrocyte processes in situ. ( A ) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB 1 D promoter. After 2 to 3 weeks, to allow for expression, Ca 2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. ( B and C ) Images of GCaMP6f-expressing WT ( Orai1 fl/fl ) (B) or Orai1 KO ( Orai1 fl/fl GFAP-Cre ) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca 2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein-coupled PARs on astrocytes and evoke Ca 2+ signaling. Movies of the Ca 2+ . ( D and E ) Summary of the Ca 2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT ( Orai1 fl/fl , black bars) and Orai1 KO ( Orail fl/fl GFAP-Cre , orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. ( F and G ) Cumulative probability plots of the amplitudes of each Ca 2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. ( H and I ) Comparison of WT and Orai1 KO Ca 2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca 2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. * P

    Techniques Used: In Situ, Expressing, Mouse Assay

    Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes. ( A ) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca 2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. ( B ) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes. ( A ) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca 2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. ( B ) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. * P

    Techniques Used: Cell Culture, Luciferase, Luminescence Assay

    Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes. ( A ) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca 2+ -free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca 2+ elicited SOCE that was significantly decreased in Orail KO ( Orai1 fl/fl nestln-Cre and Orai1 fl/fl GFAP-Cre ) cells, as measured by the rate of Ca 2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. ( B ) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca 2+ -free solution and subsequent sustained SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca 2+ -free solution followed by SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. *** P
    Figure Legend Snippet: Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes. ( A ) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca 2+ -free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca 2+ elicited SOCE that was significantly decreased in Orail KO ( Orai1 fl/fl nestln-Cre and Orai1 fl/fl GFAP-Cre ) cells, as measured by the rate of Ca 2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. ( B ) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca 2+ -free solution and subsequent sustained SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca 2+ -free solution followed by SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. *** P

    Techniques Used: Cell Culture

    Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1. ( A ) Depletion of ER Ca 2+ stores with TG (1 μM) in a Ca 2+ -free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca 2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl 3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca 2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca 2+ influx rates were calculated by measuring the initial slope of Ca 2 + entry over 18 s after readdition of 2 mM Ca 2+ solution (as shown by the dotted line for the control condition). ( B ) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca 2+ influx rates were attenuated after readdition of external Ca 2+ . The right graph shows the summary of the rate of Ca 2+ influx in WT ( Orai1 fl/+ and Orai1 fl / fl ), Orai1 HET ( Orai1 fl/+ nest,n-Cre and Orai1 fl /− ), and Orai1 KO ( Orai1 fl/fl nestin-Cre and Orai1 fl/−nestin-Cre ) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. ( C ) SOCE was abolished in cultured astrocytes from STIM1 KO mice ( STIM1 fl/fl nestin-Cre ). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. ( D ) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice ( Orai1 fl/fl GFAP-Cre ). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. ** P
    Figure Legend Snippet: Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1. ( A ) Depletion of ER Ca 2+ stores with TG (1 μM) in a Ca 2+ -free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca 2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl 3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca 2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca 2+ influx rates were calculated by measuring the initial slope of Ca 2 + entry over 18 s after readdition of 2 mM Ca 2+ solution (as shown by the dotted line for the control condition). ( B ) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca 2+ influx rates were attenuated after readdition of external Ca 2+ . The right graph shows the summary of the rate of Ca 2+ influx in WT ( Orai1 fl/+ and Orai1 fl / fl ), Orai1 HET ( Orai1 fl/+ nest,n-Cre and Orai1 fl /− ), and Orai1 KO ( Orai1 fl/fl nestin-Cre and Orai1 fl/−nestin-Cre ) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. ( C ) SOCE was abolished in cultured astrocytes from STIM1 KO mice ( STIM1 fl/fl nestin-Cre ). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. ( D ) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice ( Orai1 fl/fl GFAP-Cre ). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. ** P

    Techniques Used: Cell Culture, Mouse Assay

    Orai1 channels stimulate vesicular exocytosis after store depletion. ( A ) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 mm. ( B ) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT ( Orai1 fl/+ ) astrocyte. Scale bar, 20 μm. ( C ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. ( D ) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orail KO ( Orai1 fl/fl GFAP-Cre ) astrocyte. Scale bar, 20 μm. ( E ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. ( F ) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. ( G ) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orail KO cells, by preincubation with BAPTA-AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orail KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Orai1 channels stimulate vesicular exocytosis after store depletion. ( A ) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 mm. ( B ) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT ( Orai1 fl/+ ) astrocyte. Scale bar, 20 μm. ( C ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. ( D ) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orail KO ( Orai1 fl/fl GFAP-Cre ) astrocyte. Scale bar, 20 μm. ( E ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. ( F ) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. ( G ) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orail KO cells, by preincubation with BAPTA-AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orail KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. * P

    Techniques Used: Fluorescence

    Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes. ( A ) Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. ( B ) Left: Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. ( C and D ) Location of spH events mapped onto the footprint of an Orai1 KO ( Orai1 fl/fl GFAP-Cre ) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). ( E ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by UTP. ( F ) The average rate of UTP-evoked exocytosis per 1000 μm 2 was significantly suppressed in Orai1 KO cells, in Ca 2+ -free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca 2+ -free, n = 10 cells; TeTx, n = 7 cells. ( G ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm 2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca 2+ -free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes. ( A ) Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. ( B ) Left: Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. ( C and D ) Location of spH events mapped onto the footprint of an Orai1 KO ( Orai1 fl/fl GFAP-Cre ) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). ( E ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by UTP. ( F ) The average rate of UTP-evoked exocytosis per 1000 μm 2 was significantly suppressed in Orai1 KO cells, in Ca 2+ -free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca 2+ -free, n = 10 cells; TeTx, n = 7 cells. ( G ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm 2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca 2+ -free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. * P

    Techniques Used: Expressing

    Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells. ( A ) Administration of thrombin evokes a burst of spontaneous IPSCs on Orail (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. ( B ) sIPSC traces from the experiment in (A) shown on an expanded timescale. ( C ) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (* P = 0.02 by paired t test, n = 8 cells). ( D ) Amplitude distribution of the sIPSC events in WT slices. ( E ) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX ( n = 8 cells). ( F ) The thrombin- induced sIPSC response in CA1 neurons is abolished in Orai1 fl/fl GFAP-Cre slices. ( G ) sIPSC traces from the experiment in (F) shown on an expanded timescale. ( H ) Summary of sIPSC frequency and amplitude in Orai1 fl/fl GFAP-Cre slices before and after application of thrombin ( n = 6 cells). ( I ) Amplitude distribution of the sIPSC events in Orai1 KO slices. ( J ) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices ( n = 4 cells). Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells. ( A ) Administration of thrombin evokes a burst of spontaneous IPSCs on Orail (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. ( B ) sIPSC traces from the experiment in (A) shown on an expanded timescale. ( C ) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (* P = 0.02 by paired t test, n = 8 cells). ( D ) Amplitude distribution of the sIPSC events in WT slices. ( E ) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX ( n = 8 cells). ( F ) The thrombin- induced sIPSC response in CA1 neurons is abolished in Orai1 fl/fl GFAP-Cre slices. ( G ) sIPSC traces from the experiment in (F) shown on an expanded timescale. ( H ) Summary of sIPSC frequency and amplitude in Orai1 fl/fl GFAP-Cre slices before and after application of thrombin ( n = 6 cells). ( I ) Amplitude distribution of the sIPSC events in Orai1 KO slices. ( J ) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices ( n = 4 cells). Bar graphs show means ± SEM. * P

    Techniques Used: Patch Clamp, Isolation

    22) Product Images from "Chronic Hypoxia Increases TRPC6 Expression and Basal Intracellular Ca2+ Concentration in Rat Distal Pulmonary Venous Smooth Muscle"

    Article Title: Chronic Hypoxia Increases TRPC6 Expression and Basal Intracellular Ca2+ Concentration in Rat Distal Pulmonary Venous Smooth Muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112007

    Effects of siOrai1 transfection on basal [Ca 2+ ] i and SOCE in rat PVSMCs. A, B : Western blot showing expression of Orai1 and α-actin protein in rat PVSMC treated with siNT, siOrai1 and transfection vehicle alone (control). E, F : The changes of SOCE in siOrai1, NTsiRNA transfected PVSMCs and transfection vehicle alone treated control. *P
    Figure Legend Snippet: Effects of siOrai1 transfection on basal [Ca 2+ ] i and SOCE in rat PVSMCs. A, B : Western blot showing expression of Orai1 and α-actin protein in rat PVSMC treated with siNT, siOrai1 and transfection vehicle alone (control). E, F : The changes of SOCE in siOrai1, NTsiRNA transfected PVSMCs and transfection vehicle alone treated control. *P

    Techniques Used: Transfection, Western Blot, Expressing

    23) Product Images from "CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission"

    Article Title: CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission

    Journal: Science signaling

    doi: 10.1126/scisignal.aaw5450

    Orai1 channels generate GPCR-mediated Ca 2 +fluctuations in astrocyte processes in situ. ( A ) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB 1 D promoter. After 2 to 3 weeks, to allow for expression, Ca 2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. ( B and C ) Images of GCaMP6f-expressing WT ( Orai1 fl/fl ) (B) or Orai1 KO ( Orai1 fl/fl GFAP-Cre ) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca 2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein-coupled PARs on astrocytes and evoke Ca 2+ signaling. Movies of the Ca 2+ . ( D and E ) Summary of the Ca 2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT ( Orai1 fl/fl , black bars) and Orai1 KO ( Orail fl/fl GFAP-Cre , orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. ( F and G ) Cumulative probability plots of the amplitudes of each Ca 2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. ( H and I ) Comparison of WT and Orai1 KO Ca 2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca 2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Orai1 channels generate GPCR-mediated Ca 2 +fluctuations in astrocyte processes in situ. ( A ) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB 1 D promoter. After 2 to 3 weeks, to allow for expression, Ca 2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. ( B and C ) Images of GCaMP6f-expressing WT ( Orai1 fl/fl ) (B) or Orai1 KO ( Orai1 fl/fl GFAP-Cre ) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca 2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein-coupled PARs on astrocytes and evoke Ca 2+ signaling. Movies of the Ca 2+ . ( D and E ) Summary of the Ca 2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT ( Orai1 fl/fl , black bars) and Orai1 KO ( Orail fl/fl GFAP-Cre , orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. ( F and G ) Cumulative probability plots of the amplitudes of each Ca 2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. ( H and I ) Comparison of WT and Orai1 KO Ca 2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca 2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. * P

    Techniques Used: In Situ, Expressing, Mouse Assay

    Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes. ( A ) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca 2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. ( B ) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes. ( A ) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca 2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. ( B ) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. * P

    Techniques Used: Cell Culture, Luciferase, Luminescence Assay

    Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes. ( A ) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca 2+ -free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca 2+ elicited SOCE that was significantly decreased in Orail KO ( Orai1 fl/fl nestln-Cre and Orai1 fl/fl GFAP-Cre ) cells, as measured by the rate of Ca 2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. ( B ) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca 2+ -free solution and subsequent sustained SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca 2+ -free solution followed by SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. *** P
    Figure Legend Snippet: Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes. ( A ) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca 2+ -free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca 2+ elicited SOCE that was significantly decreased in Orail KO ( Orai1 fl/fl nestln-Cre and Orai1 fl/fl GFAP-Cre ) cells, as measured by the rate of Ca 2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. ( B ) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca 2+ -free solution and subsequent sustained SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca 2+ -free solution followed by SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. *** P

    Techniques Used: Cell Culture

    Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1. ( A ) Depletion of ER Ca 2+ stores with TG (1 μM) in a Ca 2+ -free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca 2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl 3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca 2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca 2+ influx rates were calculated by measuring the initial slope of Ca 2 + entry over 18 s after readdition of 2 mM Ca 2+ solution (as shown by the dotted line for the control condition). ( B ) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca 2+ influx rates were attenuated after readdition of external Ca 2+ . The right graph shows the summary of the rate of Ca 2+ influx in WT ( Orai1 fl/+ and Orai1 fl / fl ), Orai1 HET ( Orai1 fl/+ nest,n-Cre and Orai1 fl /− ), and Orai1 KO ( Orai1 fl/fl nestin-Cre and Orai1 fl/−nestin-Cre ) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. ( C ) SOCE was abolished in cultured astrocytes from STIM1 KO mice ( STIM1 fl/fl nestin-Cre ). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. ( D ) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice ( Orai1 fl/fl GFAP-Cre ). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. ** P
    Figure Legend Snippet: Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1. ( A ) Depletion of ER Ca 2+ stores with TG (1 μM) in a Ca 2+ -free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca 2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl 3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca 2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca 2+ influx rates were calculated by measuring the initial slope of Ca 2 + entry over 18 s after readdition of 2 mM Ca 2+ solution (as shown by the dotted line for the control condition). ( B ) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca 2+ influx rates were attenuated after readdition of external Ca 2+ . The right graph shows the summary of the rate of Ca 2+ influx in WT ( Orai1 fl/+ and Orai1 fl / fl ), Orai1 HET ( Orai1 fl/+ nest,n-Cre and Orai1 fl /− ), and Orai1 KO ( Orai1 fl/fl nestin-Cre and Orai1 fl/−nestin-Cre ) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. ( C ) SOCE was abolished in cultured astrocytes from STIM1 KO mice ( STIM1 fl/fl nestin-Cre ). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. ( D ) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice ( Orai1 fl/fl GFAP-Cre ). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. ** P

    Techniques Used: Cell Culture, Mouse Assay

    Orai1 channels stimulate vesicular exocytosis after store depletion. ( A ) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 mm. ( B ) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT ( Orai1 fl/+ ) astrocyte. Scale bar, 20 μm. ( C ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. ( D ) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orail KO ( Orai1 fl/fl GFAP-Cre ) astrocyte. Scale bar, 20 μm. ( E ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. ( F ) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. ( G ) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orail KO cells, by preincubation with BAPTA-AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orail KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Orai1 channels stimulate vesicular exocytosis after store depletion. ( A ) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 mm. ( B ) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT ( Orai1 fl/+ ) astrocyte. Scale bar, 20 μm. ( C ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. ( D ) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orail KO ( Orai1 fl/fl GFAP-Cre ) astrocyte. Scale bar, 20 μm. ( E ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. ( F ) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. ( G ) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orail KO cells, by preincubation with BAPTA-AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orail KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. * P

    Techniques Used: Fluorescence

    Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes. ( A ) Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. ( B ) Left: Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. ( C and D ) Location of spH events mapped onto the footprint of an Orai1 KO ( Orai1 fl/fl GFAP-Cre ) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). ( E ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by UTP. ( F ) The average rate of UTP-evoked exocytosis per 1000 μm 2 was significantly suppressed in Orai1 KO cells, in Ca 2+ -free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca 2+ -free, n = 10 cells; TeTx, n = 7 cells. ( G ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm 2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca 2+ -free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes. ( A ) Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. ( B ) Left: Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. ( C and D ) Location of spH events mapped onto the footprint of an Orai1 KO ( Orai1 fl/fl GFAP-Cre ) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). ( E ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by UTP. ( F ) The average rate of UTP-evoked exocytosis per 1000 μm 2 was significantly suppressed in Orai1 KO cells, in Ca 2+ -free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca 2+ -free, n = 10 cells; TeTx, n = 7 cells. ( G ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm 2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca 2+ -free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. * P

    Techniques Used: Expressing

    Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells. ( A ) Administration of thrombin evokes a burst of spontaneous IPSCs on Orail (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. ( B ) sIPSC traces from the experiment in (A) shown on an expanded timescale. ( C ) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (* P = 0.02 by paired t test, n = 8 cells). ( D ) Amplitude distribution of the sIPSC events in WT slices. ( E ) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX ( n = 8 cells). ( F ) The thrombin- induced sIPSC response in CA1 neurons is abolished in Orai1 fl/fl GFAP-Cre slices. ( G ) sIPSC traces from the experiment in (F) shown on an expanded timescale. ( H ) Summary of sIPSC frequency and amplitude in Orai1 fl/fl GFAP-Cre slices before and after application of thrombin ( n = 6 cells). ( I ) Amplitude distribution of the sIPSC events in Orai1 KO slices. ( J ) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices ( n = 4 cells). Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells. ( A ) Administration of thrombin evokes a burst of spontaneous IPSCs on Orail (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. ( B ) sIPSC traces from the experiment in (A) shown on an expanded timescale. ( C ) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (* P = 0.02 by paired t test, n = 8 cells). ( D ) Amplitude distribution of the sIPSC events in WT slices. ( E ) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX ( n = 8 cells). ( F ) The thrombin- induced sIPSC response in CA1 neurons is abolished in Orai1 fl/fl GFAP-Cre slices. ( G ) sIPSC traces from the experiment in (F) shown on an expanded timescale. ( H ) Summary of sIPSC frequency and amplitude in Orai1 fl/fl GFAP-Cre slices before and after application of thrombin ( n = 6 cells). ( I ) Amplitude distribution of the sIPSC events in Orai1 KO slices. ( J ) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices ( n = 4 cells). Bar graphs show means ± SEM. * P

    Techniques Used: Patch Clamp, Isolation

    24) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    25) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p

    Techniques Used: Expressing

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

    26) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

    27) Product Images from "KCa3.1 and TRPM7 Channels at the Uropod Regulate Migration of Activated Human T Cells"

    Article Title: KCa3.1 and TRPM7 Channels at the Uropod Regulate Migration of Activated Human T Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043859

    TRPM7 channels are crucial for T cell migration. A. Left Panel: TRPM7 gene expression is reduced by siRNA1. TRPM7 gene expression was quantified by RT-qPCR and is given in fold change relative to GAPDH expression. The data are normalized to control RNA transfected cells and correspond to mean ± SEM of 3 healthy donors each in quadruplicate. Statistical significance was determined by One Way ANOVA, while post-hoc testing was done by Holm-Sidak method. * indicates statistical significance. Right Panel: Specificity of TRPM7 siRNA. Activated T cells were transfected with TRPM7 siRNA1 for 48 hours and the gene expression for TRPM4, Orai1 and TRPM7 was quantified by RT-qPCR. The data are shown as fold change in gene expression relative to GAPDH and are normalized to control RNA transfected cells. Data are presented as mean ± SEM for 3 independent donors, with samples in quadruplicate. B. TRPM7 knockdown decreases TRPM7 currents. The average TRPM7 current was significantly decreased in TRPM7 siRNA1 transfected cells (n = 13) compared to cells transected with control RNA (n = 10). Transected cells were visualized by GFP expression. C. Effect of TRPM7 siRNA1 on T cell migration. On the left panel is reported the % of cells migr ating. The total number of cells were for control RNA 626 (n = 5 experiments; 2 donors) and for TRPM7 siRNA1 796 (n = 8 experiments; 2 donors). Right panel: T cell migration was measured in TRPM7 siRNA1 transfected cells (n = 40) and compared to control RNA transfected cells (n = 49) from 2 individuals.
    Figure Legend Snippet: TRPM7 channels are crucial for T cell migration. A. Left Panel: TRPM7 gene expression is reduced by siRNA1. TRPM7 gene expression was quantified by RT-qPCR and is given in fold change relative to GAPDH expression. The data are normalized to control RNA transfected cells and correspond to mean ± SEM of 3 healthy donors each in quadruplicate. Statistical significance was determined by One Way ANOVA, while post-hoc testing was done by Holm-Sidak method. * indicates statistical significance. Right Panel: Specificity of TRPM7 siRNA. Activated T cells were transfected with TRPM7 siRNA1 for 48 hours and the gene expression for TRPM4, Orai1 and TRPM7 was quantified by RT-qPCR. The data are shown as fold change in gene expression relative to GAPDH and are normalized to control RNA transfected cells. Data are presented as mean ± SEM for 3 independent donors, with samples in quadruplicate. B. TRPM7 knockdown decreases TRPM7 currents. The average TRPM7 current was significantly decreased in TRPM7 siRNA1 transfected cells (n = 13) compared to cells transected with control RNA (n = 10). Transected cells were visualized by GFP expression. C. Effect of TRPM7 siRNA1 on T cell migration. On the left panel is reported the % of cells migr ating. The total number of cells were for control RNA 626 (n = 5 experiments; 2 donors) and for TRPM7 siRNA1 796 (n = 8 experiments; 2 donors). Right panel: T cell migration was measured in TRPM7 siRNA1 transfected cells (n = 40) and compared to control RNA transfected cells (n = 49) from 2 individuals.

    Techniques Used: Migration, Expressing, Quantitative RT-PCR, Transfection

    Differential distribution of Orai1 and TRPM7. A. Confocal images of migrating activated T cells stained for Orai1 (green) together with either CD44 (red) or CXCR-4 (red). Yellow areas in the merge images indicate colocalization. Scale bar = 5 µm. The average correlation coefficients for the uropod (U, n = 21) and the leading-edge (L, n = 22) from 2 donors are shown in the right panel. B. Confocal images of migrating activated T cells that were fixed and stained for TRPM7 (green) together with either CD44 (red) or CXCR-4 (red). Correlation between two proteins is indicated by yellow areas in the merge images. Scale bar = 5 µm. The average correlation coefficients for the uropod (U, n = 29) and the leading-edge (L, n = 12) from 2 donors are shown in the right panel.
    Figure Legend Snippet: Differential distribution of Orai1 and TRPM7. A. Confocal images of migrating activated T cells stained for Orai1 (green) together with either CD44 (red) or CXCR-4 (red). Yellow areas in the merge images indicate colocalization. Scale bar = 5 µm. The average correlation coefficients for the uropod (U, n = 21) and the leading-edge (L, n = 22) from 2 donors are shown in the right panel. B. Confocal images of migrating activated T cells that were fixed and stained for TRPM7 (green) together with either CD44 (red) or CXCR-4 (red). Correlation between two proteins is indicated by yellow areas in the merge images. Scale bar = 5 µm. The average correlation coefficients for the uropod (U, n = 29) and the leading-edge (L, n = 12) from 2 donors are shown in the right panel.

    Techniques Used: Staining

    28) Product Images from "CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission"

    Article Title: CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission

    Journal: Science signaling

    doi: 10.1126/scisignal.aaw5450

    Orai1 channels generate GPCR-mediated Ca 2 +fluctuations in astrocyte processes in situ. ( A ) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB 1 D promoter. After 2 to 3 weeks, to allow for expression, Ca 2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. ( B and C ) Images of GCaMP6f-expressing WT ( Orai1 fl/fl ) (B) or Orai1 KO ( Orai1 fl/fl GFAP-Cre ) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca 2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein-coupled PARs on astrocytes and evoke Ca 2+ signaling. Movies of the Ca 2+ . ( D and E ) Summary of the Ca 2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT ( Orai1 fl/fl , black bars) and Orai1 KO ( Orail fl/fl GFAP-Cre , orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. ( F and G ) Cumulative probability plots of the amplitudes of each Ca 2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. ( H and I ) Comparison of WT and Orai1 KO Ca 2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca 2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Orai1 channels generate GPCR-mediated Ca 2 +fluctuations in astrocyte processes in situ. ( A ) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB 1 D promoter. After 2 to 3 weeks, to allow for expression, Ca 2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. ( B and C ) Images of GCaMP6f-expressing WT ( Orai1 fl/fl ) (B) or Orai1 KO ( Orai1 fl/fl GFAP-Cre ) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca 2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein-coupled PARs on astrocytes and evoke Ca 2+ signaling. Movies of the Ca 2+ . ( D and E ) Summary of the Ca 2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT ( Orai1 fl/fl , black bars) and Orai1 KO ( Orail fl/fl GFAP-Cre , orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. ( F and G ) Cumulative probability plots of the amplitudes of each Ca 2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. ( H and I ) Comparison of WT and Orai1 KO Ca 2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca 2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. * P

    Techniques Used: In Situ, Expressing, Mouse Assay

    Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes. ( A ) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca 2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. ( B ) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes. ( A ) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca 2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. ( B ) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. * P

    Techniques Used: Cell Culture, Luciferase, Luminescence Assay

    Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes. ( A ) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca 2+ -free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca 2+ elicited SOCE that was significantly decreased in Orail KO ( Orai1 fl/fl nestln-Cre and Orai1 fl/fl GFAP-Cre ) cells, as measured by the rate of Ca 2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. ( B ) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca 2+ -free solution and subsequent sustained SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca 2+ -free solution followed by SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. *** P
    Figure Legend Snippet: Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes. ( A ) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca 2+ -free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca 2+ elicited SOCE that was significantly decreased in Orail KO ( Orai1 fl/fl nestln-Cre and Orai1 fl/fl GFAP-Cre ) cells, as measured by the rate of Ca 2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. ( B ) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca 2+ -free solution and subsequent sustained SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca 2+ -free solution followed by SOCE in 2 mM Ca 2+ solution. SOCE was significantly attenuated in Orail KO ( Orai1 fl/fl GFAP-Cre ) and STIM1 KO ( STIM1 fl/fl nestin-Cre ) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. *** P

    Techniques Used: Cell Culture

    Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1. ( A ) Depletion of ER Ca 2+ stores with TG (1 μM) in a Ca 2+ -free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca 2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl 3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca 2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca 2+ influx rates were calculated by measuring the initial slope of Ca 2 + entry over 18 s after readdition of 2 mM Ca 2+ solution (as shown by the dotted line for the control condition). ( B ) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca 2+ influx rates were attenuated after readdition of external Ca 2+ . The right graph shows the summary of the rate of Ca 2+ influx in WT ( Orai1 fl/+ and Orai1 fl / fl ), Orai1 HET ( Orai1 fl/+ nest,n-Cre and Orai1 fl /− ), and Orai1 KO ( Orai1 fl/fl nestin-Cre and Orai1 fl/−nestin-Cre ) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. ( C ) SOCE was abolished in cultured astrocytes from STIM1 KO mice ( STIM1 fl/fl nestin-Cre ). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. ( D ) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice ( Orai1 fl/fl GFAP-Cre ). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. ** P
    Figure Legend Snippet: Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1. ( A ) Depletion of ER Ca 2+ stores with TG (1 μM) in a Ca 2+ -free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca 2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl 3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca 2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca 2+ influx rates were calculated by measuring the initial slope of Ca 2 + entry over 18 s after readdition of 2 mM Ca 2+ solution (as shown by the dotted line for the control condition). ( B ) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca 2+ influx rates were attenuated after readdition of external Ca 2+ . The right graph shows the summary of the rate of Ca 2+ influx in WT ( Orai1 fl/+ and Orai1 fl / fl ), Orai1 HET ( Orai1 fl/+ nest,n-Cre and Orai1 fl /− ), and Orai1 KO ( Orai1 fl/fl nestin-Cre and Orai1 fl/−nestin-Cre ) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. ( C ) SOCE was abolished in cultured astrocytes from STIM1 KO mice ( STIM1 fl/fl nestin-Cre ). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. ( D ) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice ( Orai1 fl/fl GFAP-Cre ). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. ** P

    Techniques Used: Cell Culture, Mouse Assay

    Orai1 channels stimulate vesicular exocytosis after store depletion. ( A ) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 mm. ( B ) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT ( Orai1 fl/+ ) astrocyte. Scale bar, 20 μm. ( C ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. ( D ) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orail KO ( Orai1 fl/fl GFAP-Cre ) astrocyte. Scale bar, 20 μm. ( E ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. ( F ) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. ( G ) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orail KO cells, by preincubation with BAPTA-AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orail KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Orai1 channels stimulate vesicular exocytosis after store depletion. ( A ) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 mm. ( B ) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT ( Orai1 fl/+ ) astrocyte. Scale bar, 20 μm. ( C ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. ( D ) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orail KO ( Orai1 fl/fl GFAP-Cre ) astrocyte. Scale bar, 20 μm. ( E ) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. ( F ) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. ( G ) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orail KO cells, by preincubation with BAPTA-AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orail KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. * P

    Techniques Used: Fluorescence

    Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes. ( A ) Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. ( B ) Left: Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. ( C and D ) Location of spH events mapped onto the footprint of an Orai1 KO ( Orai1 fl/fl GFAP-Cre ) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). ( E ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by UTP. ( F ) The average rate of UTP-evoked exocytosis per 1000 μm 2 was significantly suppressed in Orai1 KO cells, in Ca 2+ -free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca 2+ -free, n = 10 cells; TeTx, n = 7 cells. ( G ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm 2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca 2+ -free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes. ( A ) Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. ( B ) Left: Location of spH events (blue dots) mapped onto the footprint of a WT ( Orai1 fl/+ ) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. ( C and D ) Location of spH events mapped onto the footprint of an Orai1 KO ( Orai1 fl/fl GFAP-Cre ) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). ( E ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by UTP. ( F ) The average rate of UTP-evoked exocytosis per 1000 μm 2 was significantly suppressed in Orai1 KO cells, in Ca 2+ -free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca 2+ -free, n = 10 cells; TeTx, n = 7 cells. ( G ) Cumulative event plots for WT, Orai1 KO, Ca 2+ -free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm 2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca 2+ -free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. * P

    Techniques Used: Expressing

    Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells. ( A ) Administration of thrombin evokes a burst of spontaneous IPSCs on Orail (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. ( B ) sIPSC traces from the experiment in (A) shown on an expanded timescale. ( C ) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (* P = 0.02 by paired t test, n = 8 cells). ( D ) Amplitude distribution of the sIPSC events in WT slices. ( E ) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX ( n = 8 cells). ( F ) The thrombin- induced sIPSC response in CA1 neurons is abolished in Orai1 fl/fl GFAP-Cre slices. ( G ) sIPSC traces from the experiment in (F) shown on an expanded timescale. ( H ) Summary of sIPSC frequency and amplitude in Orai1 fl/fl GFAP-Cre slices before and after application of thrombin ( n = 6 cells). ( I ) Amplitude distribution of the sIPSC events in Orai1 KO slices. ( J ) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices ( n = 4 cells). Bar graphs show means ± SEM. * P
    Figure Legend Snippet: Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells. ( A ) Administration of thrombin evokes a burst of spontaneous IPSCs on Orail (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. ( B ) sIPSC traces from the experiment in (A) shown on an expanded timescale. ( C ) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (* P = 0.02 by paired t test, n = 8 cells). ( D ) Amplitude distribution of the sIPSC events in WT slices. ( E ) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX ( n = 8 cells). ( F ) The thrombin- induced sIPSC response in CA1 neurons is abolished in Orai1 fl/fl GFAP-Cre slices. ( G ) sIPSC traces from the experiment in (F) shown on an expanded timescale. ( H ) Summary of sIPSC frequency and amplitude in Orai1 fl/fl GFAP-Cre slices before and after application of thrombin ( n = 6 cells). ( I ) Amplitude distribution of the sIPSC events in Orai1 KO slices. ( J ) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices ( n = 4 cells). Bar graphs show means ± SEM. * P

    Techniques Used: Patch Clamp, Isolation

    29) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p

    Techniques Used: Expressing

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

    30) Product Images from "Orai/CRACM1 and KCa3.1 ion channels interact in the human lung mast cell plasma membrane"

    Article Title: Orai/CRACM1 and KCa3.1 ion channels interact in the human lung mast cell plasma membrane

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-015-0112-z

    Orai2 and K Ca 3.1 proteins do not co-immunoprecipitate under the conditions used to co-immunoprecipitate Orai1 and K Ca 3.1. a Western blots using either an antibody recognising the myc epitope (left) or an antibody recognising the FLAG epitope (right) of HEK293 cell lysates. Lysates expressed either myc epitope tagged Orai2, FLAG epitope-tagged K Ca 3.1, or both as indicated in the panel above. b HEK293 cell lysates expressing the proteins indicated in the panel above were immunoprecipitated with an anti-myc antibody. Immunoprecipitates were then Western blotted using either an anti-Orai2 antibody (left) or an anti-FLAG antibody (right). Control HEK293 cell lysate expressing K Ca 3.1-FLAG protein. c As ( b ) except cell lysates were immunoprecipitated with an anti-FLAG antibody and then Western blotted with an anti-Orai2 antibody (left) or an anti-FLAG antibody (right). Control HEK293 cell lysate expressing Orai2-myc protein. Blots shown are representative of 3 independent experiments
    Figure Legend Snippet: Orai2 and K Ca 3.1 proteins do not co-immunoprecipitate under the conditions used to co-immunoprecipitate Orai1 and K Ca 3.1. a Western blots using either an antibody recognising the myc epitope (left) or an antibody recognising the FLAG epitope (right) of HEK293 cell lysates. Lysates expressed either myc epitope tagged Orai2, FLAG epitope-tagged K Ca 3.1, or both as indicated in the panel above. b HEK293 cell lysates expressing the proteins indicated in the panel above were immunoprecipitated with an anti-myc antibody. Immunoprecipitates were then Western blotted using either an anti-Orai2 antibody (left) or an anti-FLAG antibody (right). Control HEK293 cell lysate expressing K Ca 3.1-FLAG protein. c As ( b ) except cell lysates were immunoprecipitated with an anti-FLAG antibody and then Western blotted with an anti-Orai2 antibody (left) or an anti-FLAG antibody (right). Control HEK293 cell lysate expressing Orai2-myc protein. Blots shown are representative of 3 independent experiments

    Techniques Used: Western Blot, FLAG-tag, Expressing, Immunoprecipitation

    Orai1 and K Ca 3.1 co-localise in the plasma membrane. a HEK293 cells, dually transfected with FLAG-tagged K Ca 3.1 and myc-tagged Orai1 and then immunostained, show co-localisation in the plasma membrane by single plane confocal microscopy (top panels). Dually transfected HEK293 show negative staining for appropriate isotype controls (bottom panels): rabbit IgG control, dual stained with anti-myc, and mouse IgG1 control dual stained with anti-FLAG. b Fluorescence intensity plot shows increased fluorescence at the plasma membrane. myc-Orai1 is shown in green and FLAG-K Ca 3.1 in red. Arrows indicate increased fluorescence where the region of interest (ROI) intersects the plasma membrane. c HEK293 cells, dually transfected with FLAG-tagged K Ca 3.1 and myc-tagged Orai2 and then immunostained, show poor co-localisation in the plasma membrane by single plane confocal microscopy (top panels). Dually transfected HEK293 show negative staining for appropriate isotype controls (bottom panels): rabbit IgG control, dual stained with anti-myc, and mouse IgG1 control dual stained with anti-FLAG. d Fluorescence intensity plot shows poor co-localisation of K Ca 3.1 and Orai2 signals. myc-Orai2 is shown in green and FLAG-K Ca 3.1 in red. Scale bars are 10 μm
    Figure Legend Snippet: Orai1 and K Ca 3.1 co-localise in the plasma membrane. a HEK293 cells, dually transfected with FLAG-tagged K Ca 3.1 and myc-tagged Orai1 and then immunostained, show co-localisation in the plasma membrane by single plane confocal microscopy (top panels). Dually transfected HEK293 show negative staining for appropriate isotype controls (bottom panels): rabbit IgG control, dual stained with anti-myc, and mouse IgG1 control dual stained with anti-FLAG. b Fluorescence intensity plot shows increased fluorescence at the plasma membrane. myc-Orai1 is shown in green and FLAG-K Ca 3.1 in red. Arrows indicate increased fluorescence where the region of interest (ROI) intersects the plasma membrane. c HEK293 cells, dually transfected with FLAG-tagged K Ca 3.1 and myc-tagged Orai2 and then immunostained, show poor co-localisation in the plasma membrane by single plane confocal microscopy (top panels). Dually transfected HEK293 show negative staining for appropriate isotype controls (bottom panels): rabbit IgG control, dual stained with anti-myc, and mouse IgG1 control dual stained with anti-FLAG. d Fluorescence intensity plot shows poor co-localisation of K Ca 3.1 and Orai2 signals. myc-Orai2 is shown in green and FLAG-K Ca 3.1 in red. Scale bars are 10 μm

    Techniques Used: Transfection, Confocal Microscopy, Negative Staining, Staining, Fluorescence

    Orai1 and K Ca 3.1 proteins co-immunoprecipitate. a Western blots using either an antibody recognising the myc epitope (left) or an antibody recognising the FLAG epitope (right) of HEK293 cell lysates. Lysates expressed either myc epitope tagged Orai1, FLAG epitope-tagged K Ca 3.1, or both as indicated in the panel above. b Lysates of HEK293 cells expressing the indicated proteins were immunoprecipitated with an anti-myc antibody. Immunoprecipitates were then Western blotted using either an anti-Orai1 antibody (left) or an anti-FLAG antibody (right). c As ( b ) except cell lysates were immunoprecipitated with an anti-FLAG antibody and then Western blotted with an anti-FLAG antibody (left) or an anti-myc antibody (right). Blots shown are representative of 3 independent experiments
    Figure Legend Snippet: Orai1 and K Ca 3.1 proteins co-immunoprecipitate. a Western blots using either an antibody recognising the myc epitope (left) or an antibody recognising the FLAG epitope (right) of HEK293 cell lysates. Lysates expressed either myc epitope tagged Orai1, FLAG epitope-tagged K Ca 3.1, or both as indicated in the panel above. b Lysates of HEK293 cells expressing the indicated proteins were immunoprecipitated with an anti-myc antibody. Immunoprecipitates were then Western blotted using either an anti-Orai1 antibody (left) or an anti-FLAG antibody (right). c As ( b ) except cell lysates were immunoprecipitated with an anti-FLAG antibody and then Western blotted with an anti-FLAG antibody (left) or an anti-myc antibody (right). Blots shown are representative of 3 independent experiments

    Techniques Used: Western Blot, FLAG-tag, Expressing, Immunoprecipitation

    FcεRI- and 1-EBIO-dependent HLMC K Ca 3.1 currents are inhibited by expression of an Orai1-E106Q dominant-negative mutant. Transduction of HLMCs with an Orai1-E106Q dominant-negative mutant ablated ( a ) 1-EBIO- dependent and ( b ) FcεRI-dependent K Ca 3.1 currents. For clarity, data are presented as the subtracted net activation-dependent currents (activation minus baseline) for each condition, expressed as mean ± SEM
    Figure Legend Snippet: FcεRI- and 1-EBIO-dependent HLMC K Ca 3.1 currents are inhibited by expression of an Orai1-E106Q dominant-negative mutant. Transduction of HLMCs with an Orai1-E106Q dominant-negative mutant ablated ( a ) 1-EBIO- dependent and ( b ) FcεRI-dependent K Ca 3.1 currents. For clarity, data are presented as the subtracted net activation-dependent currents (activation minus baseline) for each condition, expressed as mean ± SEM

    Techniques Used: Expressing, Dominant Negative Mutation, Transduction, Activation Assay

    31) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

    32) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p

    Techniques Used: Expressing

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

    33) Product Images from "Inhibition of Polyamine Biosynthesis Reverses Ca2+ Channel Remodeling in Colon Cancer Cells"

    Article Title: Inhibition of Polyamine Biosynthesis Reverses Ca2+ Channel Remodeling in Colon Cancer Cells

    Journal: Cancers

    doi: 10.3390/cancers11010083

    Effects of DFMO on the expression of proteins involved in SOCE in colon cancer HT29 cells. HT29 cells were treated with vehicle (control) or DFMO 5 mM, and then cells were lysed and subjected to Western blotting with antibodies against TRPC1, STIM1, STIM2, ORAI1, ORAI2 and ORAI3, followed by reprobing with anti-β-actin antibody for protein loading control. Bar graphs represent specific protein expression normalized to the β-actin content. Data are from n = 3 experiments (* p
    Figure Legend Snippet: Effects of DFMO on the expression of proteins involved in SOCE in colon cancer HT29 cells. HT29 cells were treated with vehicle (control) or DFMO 5 mM, and then cells were lysed and subjected to Western blotting with antibodies against TRPC1, STIM1, STIM2, ORAI1, ORAI2 and ORAI3, followed by reprobing with anti-β-actin antibody for protein loading control. Bar graphs represent specific protein expression normalized to the β-actin content. Data are from n = 3 experiments (* p

    Techniques Used: Expressing, Western Blot

    Effects of DFMO on the expression of genes coding for SOCE molecular players in HT29 cells. mRNA expression levels of selected genes were determined using qRT-PCR of extracts from control and DFMO-treated HT29 cells. β-actin was used as a reference. Data results are mean ± SEM from DFMO-treated cells relative to untreated cells Data are from n = 7, 7, 6, 6, 6, and 5 experiments for TRPC1 , STIM1 , STIM2 , ORAI1 , ORAI2 , and ORAI3 , respectively * p
    Figure Legend Snippet: Effects of DFMO on the expression of genes coding for SOCE molecular players in HT29 cells. mRNA expression levels of selected genes were determined using qRT-PCR of extracts from control and DFMO-treated HT29 cells. β-actin was used as a reference. Data results are mean ± SEM from DFMO-treated cells relative to untreated cells Data are from n = 7, 7, 6, 6, 6, and 5 experiments for TRPC1 , STIM1 , STIM2 , ORAI1 , ORAI2 , and ORAI3 , respectively * p

    Techniques Used: Expressing, Quantitative RT-PCR

    34) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    35) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

    36) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p

    Techniques Used: Expressing

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

    37) Product Images from "Inhibition of Polyamine Biosynthesis Reverses Ca2+ Channel Remodeling in Colon Cancer Cells"

    Article Title: Inhibition of Polyamine Biosynthesis Reverses Ca2+ Channel Remodeling in Colon Cancer Cells

    Journal: Cancers

    doi: 10.3390/cancers11010083

    Effects of DFMO on the expression of proteins involved in SOCE in colon cancer HT29 cells. HT29 cells were treated with vehicle (control) or DFMO 5 mM, and then cells were lysed and subjected to Western blotting with antibodies against TRPC1, STIM1, STIM2, ORAI1, ORAI2 and ORAI3, followed by reprobing with anti-β-actin antibody for protein loading control. Bar graphs represent specific protein expression normalized to the β-actin content. Data are from n = 3 experiments (* p
    Figure Legend Snippet: Effects of DFMO on the expression of proteins involved in SOCE in colon cancer HT29 cells. HT29 cells were treated with vehicle (control) or DFMO 5 mM, and then cells were lysed and subjected to Western blotting with antibodies against TRPC1, STIM1, STIM2, ORAI1, ORAI2 and ORAI3, followed by reprobing with anti-β-actin antibody for protein loading control. Bar graphs represent specific protein expression normalized to the β-actin content. Data are from n = 3 experiments (* p

    Techniques Used: Expressing, Western Blot

    Effects of DFMO on the expression of genes coding for SOCE molecular players in HT29 cells. mRNA expression levels of selected genes were determined using qRT-PCR of extracts from control and DFMO-treated HT29 cells. β-actin was used as a reference. Data results are mean ± SEM from DFMO-treated cells relative to untreated cells Data are from n = 7, 7, 6, 6, 6, and 5 experiments for TRPC1 , STIM1 , STIM2 , ORAI1 , ORAI2 , and ORAI3 , respectively * p
    Figure Legend Snippet: Effects of DFMO on the expression of genes coding for SOCE molecular players in HT29 cells. mRNA expression levels of selected genes were determined using qRT-PCR of extracts from control and DFMO-treated HT29 cells. β-actin was used as a reference. Data results are mean ± SEM from DFMO-treated cells relative to untreated cells Data are from n = 7, 7, 6, 6, 6, and 5 experiments for TRPC1 , STIM1 , STIM2 , ORAI1 , ORAI2 , and ORAI3 , respectively * p

    Techniques Used: Expressing, Quantitative RT-PCR

    38) Product Images from "Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle"

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    Journal: The European respiratory journal

    doi: 10.1183/09031936.00090511

    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Figure Legend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Techniques Used: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM
    Figure Legend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Techniques Used: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within
    Figure Legend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Techniques Used: Staining, Confocal Microscopy, Expressing

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    Alomone Labs orai1
    Effect of <t>Orai1</t> small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth
    Orai1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/orai1/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    orai1 - by Bioz Stars, 2022-01
    92/100 stars
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    93
    Alomone Labs anti orai1
    Mechanisms of SOCE in optic nerve glia. ATP-mediated calcium signalling in optic nerve glia is via P2Y G-protein-coupled receptors and the formation of IP3, which acts on IP3R1 on the ER to trigger release of Ca 2+ into the cytosol. Subsequent replenishment of ER stores in astrocytes and oligodendrocytes is dependent on SOCE via TRPM3 and <t>Orai1,</t> which form the plasmalemmal Ca 2+ channels, and mainly Stim1, which acts as the sensor of Ca 2+ depletion, and uptake into the ER is via SERCA pumps. Oligodendrocytes also express Stim2, which may be localized to the myelin, whereas Orai1, Stim1 and TRPM3 are localized to oligodendroglial somata. Notably, calcium homeostasis in optic nerve glia depends on an apparent continuous Ca 2+ influx from the extracellular milieu that is largely dependent on SOCE. Moreover, SOCE is essential for the sustainability of ATP-mediated Ca 2+ signalling in optic nerve glia, which has a central role in white matter physiology and pathology
    Anti Orai1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti orai1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti orai1 - by Bioz Stars, 2022-01
    93/100 stars
      Buy from Supplier

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    Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Journal: The European respiratory journal

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    doi: 10.1183/09031936.00090511

    Figure Lengend Snippet: Effect of Orai1 small interference RNA (siRNA) on intracellular Ca 2+ ([Ca 2+ ] i ) and store-operated Ca 2+ entry (SOCE) in human airway smooth muscle (ASM) cells. a) Transfection efficiency of Orai1 using siRNA was verified by Western blot analysis. Smooth

    Article Snippet: Liao Y, Erxleben C, Abramowitz J, et al. Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels.

    Techniques: Transfection, Western Blot

    Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Journal: The European respiratory journal

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    doi: 10.1183/09031936.00090511

    Figure Lengend Snippet: Effect of tumour necrosis factor (TNF)-α and caveolin (Cav)-1 on stromal interaction molecule (STIM)1 and Orai1 expression in human airway smooth muscle (ASM) cells. a) Exposure to TNF-α increased both STIM1 and Orai1 expression in ASM

    Article Snippet: Liao Y, Erxleben C, Abramowitz J, et al. Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels.

    Techniques: Expressing

    Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Journal: The European respiratory journal

    Article Title: Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle

    doi: 10.1183/09031936.00090511

    Figure Lengend Snippet: Localisation of caveolin (Cav)-1, stromal interaction molecule (STIM)1 and Orai1 in human airway smooth muscle (ASM) cells. a) Immunocytochemical staining of human ASM cells and confocal microscopy demonstrated expression of both Cav-1 and Orai1 within

    Article Snippet: Liao Y, Erxleben C, Abramowitz J, et al. Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels.

    Techniques: Staining, Confocal Microscopy, Expressing

    Mechanisms of SOCE in optic nerve glia. ATP-mediated calcium signalling in optic nerve glia is via P2Y G-protein-coupled receptors and the formation of IP3, which acts on IP3R1 on the ER to trigger release of Ca 2+ into the cytosol. Subsequent replenishment of ER stores in astrocytes and oligodendrocytes is dependent on SOCE via TRPM3 and Orai1, which form the plasmalemmal Ca 2+ channels, and mainly Stim1, which acts as the sensor of Ca 2+ depletion, and uptake into the ER is via SERCA pumps. Oligodendrocytes also express Stim2, which may be localized to the myelin, whereas Orai1, Stim1 and TRPM3 are localized to oligodendroglial somata. Notably, calcium homeostasis in optic nerve glia depends on an apparent continuous Ca 2+ influx from the extracellular milieu that is largely dependent on SOCE. Moreover, SOCE is essential for the sustainability of ATP-mediated Ca 2+ signalling in optic nerve glia, which has a central role in white matter physiology and pathology

    Journal: Brain Structure & Function

    Article Title: Store-operated calcium entry is essential for glial calcium signalling in CNS white matter

    doi: 10.1007/s00429-017-1380-8

    Figure Lengend Snippet: Mechanisms of SOCE in optic nerve glia. ATP-mediated calcium signalling in optic nerve glia is via P2Y G-protein-coupled receptors and the formation of IP3, which acts on IP3R1 on the ER to trigger release of Ca 2+ into the cytosol. Subsequent replenishment of ER stores in astrocytes and oligodendrocytes is dependent on SOCE via TRPM3 and Orai1, which form the plasmalemmal Ca 2+ channels, and mainly Stim1, which acts as the sensor of Ca 2+ depletion, and uptake into the ER is via SERCA pumps. Oligodendrocytes also express Stim2, which may be localized to the myelin, whereas Orai1, Stim1 and TRPM3 are localized to oligodendroglial somata. Notably, calcium homeostasis in optic nerve glia depends on an apparent continuous Ca 2+ influx from the extracellular milieu that is largely dependent on SOCE. Moreover, SOCE is essential for the sustainability of ATP-mediated Ca 2+ signalling in optic nerve glia, which has a central role in white matter physiology and pathology

    Article Snippet: Primary antibodies were diluted in blocking solution and tissues/cells incubated overnight at 4 °C; anti-STIM1, anti-STIM2, anti-ORAI1, anti-TRPM3 were raised in rabbits (Alomone) and used at 1:300; chicken anti-GFAP (Chemicon) was used at 1:500.

    Techniques:

    Expression of Orai/Stim transcripts in the mouse optic nerve. qRT-PCR was performed on lysates of acutely isolated optic nerves from WT postnatal mice (aged P9–P12) and adult mice (P30–P40). Data are from ten pooled optic nerves in each age group, run in triplicate, expressed as relative mRNA levels (2 -ΔCt ) compared to the housekeeping gene GAPDH (mean ± SEM, n = 3). Orai1 was the most highly expressed Orai isoform in the postnatal nerve (* p

    Journal: Brain Structure & Function

    Article Title: Store-operated calcium entry is essential for glial calcium signalling in CNS white matter

    doi: 10.1007/s00429-017-1380-8

    Figure Lengend Snippet: Expression of Orai/Stim transcripts in the mouse optic nerve. qRT-PCR was performed on lysates of acutely isolated optic nerves from WT postnatal mice (aged P9–P12) and adult mice (P30–P40). Data are from ten pooled optic nerves in each age group, run in triplicate, expressed as relative mRNA levels (2 -ΔCt ) compared to the housekeeping gene GAPDH (mean ± SEM, n = 3). Orai1 was the most highly expressed Orai isoform in the postnatal nerve (* p

    Article Snippet: Primary antibodies were diluted in blocking solution and tissues/cells incubated overnight at 4 °C; anti-STIM1, anti-STIM2, anti-ORAI1, anti-TRPM3 were raised in rabbits (Alomone) and used at 1:300; chicken anti-GFAP (Chemicon) was used at 1:500.

    Techniques: Expressing, Quantitative RT-PCR, Isolation, Mouse Assay

    Expression of Orai/Stim in optic nerve oligodendrocytes. Immunolabelling for Orai1 ( a , green ), Stim1 ( b , green ) and Stim2 ( c , green ) in PLP-DsRed mice to identify oligodendrocytes ( a – c , red ), in optic nerve sections ( ai – iv, bi – iv, ci – iv ) and explant cultures ( av, bv, cv ). Confocal micrographs illustrate single channels ( ai, aii, bi, bii, ci, cii ) and merged cannels ( aiii, biii, ciii ). Expression of Orai1 and Stim1 is localized to oligodendroglial somata, whereas Stim2 immunostaining was primarily within the fascicles of myelinated axons, as demonstrated by the colocalization channels, illustrating voxels in which green and red channels are of equal intensity and appear yellow ( aiv, biv, civ ). Oligodendrocytes in explant cultures are immunopositive for Orai1 ( av ), Stim1 ( bv ) and Stim2 ( cv ). No immunostaining was observed in negative controls that were pre-incubated in blocking peptides for Orai1 ( inset , aiii ), Stim1 ( inset , biii ) and Stim2 ( inset , ciii ). Nuclei are stained with Hoechst blue. Scale bars a, b 10 µm, c 20 μm

    Journal: Brain Structure & Function

    Article Title: Store-operated calcium entry is essential for glial calcium signalling in CNS white matter

    doi: 10.1007/s00429-017-1380-8

    Figure Lengend Snippet: Expression of Orai/Stim in optic nerve oligodendrocytes. Immunolabelling for Orai1 ( a , green ), Stim1 ( b , green ) and Stim2 ( c , green ) in PLP-DsRed mice to identify oligodendrocytes ( a – c , red ), in optic nerve sections ( ai – iv, bi – iv, ci – iv ) and explant cultures ( av, bv, cv ). Confocal micrographs illustrate single channels ( ai, aii, bi, bii, ci, cii ) and merged cannels ( aiii, biii, ciii ). Expression of Orai1 and Stim1 is localized to oligodendroglial somata, whereas Stim2 immunostaining was primarily within the fascicles of myelinated axons, as demonstrated by the colocalization channels, illustrating voxels in which green and red channels are of equal intensity and appear yellow ( aiv, biv, civ ). Oligodendrocytes in explant cultures are immunopositive for Orai1 ( av ), Stim1 ( bv ) and Stim2 ( cv ). No immunostaining was observed in negative controls that were pre-incubated in blocking peptides for Orai1 ( inset , aiii ), Stim1 ( inset , biii ) and Stim2 ( inset , ciii ). Nuclei are stained with Hoechst blue. Scale bars a, b 10 µm, c 20 μm

    Article Snippet: Primary antibodies were diluted in blocking solution and tissues/cells incubated overnight at 4 °C; anti-STIM1, anti-STIM2, anti-ORAI1, anti-TRPM3 were raised in rabbits (Alomone) and used at 1:300; chicken anti-GFAP (Chemicon) was used at 1:500.

    Techniques: Expressing, Plasmid Purification, Mouse Assay, Immunostaining, Incubation, Blocking Assay, Staining

    Expression of Orai/Stim in optic nerve astrocytes. Double immunofluorescence labelling for Orai1 ( a , green ), Stim1 ( b , green ) and Stim2 ( c , green ), with GFAP to identify astrocytes ( a – c , red ), in WT optic nerve sections ( ai – iv , bi– iv, ci – iv ) and explant cultures ( av, bv, cv ). Confocal micrographs illustrate single channels ( ai, aii, bi, bii, ci, cii ) and merged cannels ( aiii, biii, ciii ). Expression of Orai1 and Stim1 is localized to astrocyte processes, whereas astrocytes were immunonegative for Stim2, as demonstrated by the colocalization channels, illustrating voxels in which green and red channels are of equal intensity and appear yellow ( aiv, biv, civ ). Astrocytes in explant cultures are immunopositive for Orai1 ( av ) and Stim1 ( bv ), but are immunonegative for Stim2 ( cv ). No immunostaining was observed in negative controls that were pre-incubated in blocking peptides for Orai1 ( inset , aiii ), Stim1 ( inset , biii ) and Stim2 ( inset , ciii ). Nuclei are stained with Hoechst blue. Scale bars a, b 10 µm, c 20 μm

    Journal: Brain Structure & Function

    Article Title: Store-operated calcium entry is essential for glial calcium signalling in CNS white matter

    doi: 10.1007/s00429-017-1380-8

    Figure Lengend Snippet: Expression of Orai/Stim in optic nerve astrocytes. Double immunofluorescence labelling for Orai1 ( a , green ), Stim1 ( b , green ) and Stim2 ( c , green ), with GFAP to identify astrocytes ( a – c , red ), in WT optic nerve sections ( ai – iv , bi– iv, ci – iv ) and explant cultures ( av, bv, cv ). Confocal micrographs illustrate single channels ( ai, aii, bi, bii, ci, cii ) and merged cannels ( aiii, biii, ciii ). Expression of Orai1 and Stim1 is localized to astrocyte processes, whereas astrocytes were immunonegative for Stim2, as demonstrated by the colocalization channels, illustrating voxels in which green and red channels are of equal intensity and appear yellow ( aiv, biv, civ ). Astrocytes in explant cultures are immunopositive for Orai1 ( av ) and Stim1 ( bv ), but are immunonegative for Stim2 ( cv ). No immunostaining was observed in negative controls that were pre-incubated in blocking peptides for Orai1 ( inset , aiii ), Stim1 ( inset , biii ) and Stim2 ( inset , ciii ). Nuclei are stained with Hoechst blue. Scale bars a, b 10 µm, c 20 μm

    Article Snippet: Primary antibodies were diluted in blocking solution and tissues/cells incubated overnight at 4 °C; anti-STIM1, anti-STIM2, anti-ORAI1, anti-TRPM3 were raised in rabbits (Alomone) and used at 1:300; chicken anti-GFAP (Chemicon) was used at 1:500.

    Techniques: Expressing, Immunofluorescence, Immunostaining, Incubation, Blocking Assay, Staining