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    Structured Review

    Alomone Labs orai1
    siRNA knockdown of Stim1 , <t>Orai1</t> and Trpc1 and gene silencing effects on SOCE. ( A and B ) mRNA levels for Stim1 , Orai1 and Trpc1 upon knockdown (KO) of each gene separately as well as upon the simultaneous silencing of Stim1 and Orai1 ; ( C ) traces of average [Ca 2+ ] i in response to an elevation of extracellular Ca 2+ from 0 to 2.6 mM. Cells were treated with thapsigargin (1 µM) during 10 min prior to the reintroduction of a Ca 2+ -containing solution (not shown); ( D ) Average Δpeak [Ca 2+ ] i increase at different time points under the conditions shown in C . ( E ) Percentage remaining Δpeak [Ca 2+ ] i at 7 min under the different KO conditions compared with control (set to 100%). The difference in [Ca 2+ ] i levels was analysed by two-way repeated measure ANOVA with Dunnett's multiple comparisons test at each time point. *P
    Orai1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Extracellular ATP activates store-operated Ca2+ entry in white adipocytes: functional evidence for STIM1 and ORAI1"

    Article Title: Extracellular ATP activates store-operated Ca2+ entry in white adipocytes: functional evidence for STIM1 and ORAI1

    Journal: Biochemical Journal

    doi: 10.1042/BCJ20170484

    siRNA knockdown of Stim1 , Orai1 and Trpc1 and gene silencing effects on SOCE. ( A and B ) mRNA levels for Stim1 , Orai1 and Trpc1 upon knockdown (KO) of each gene separately as well as upon the simultaneous silencing of Stim1 and Orai1 ; ( C ) traces of average [Ca 2+ ] i in response to an elevation of extracellular Ca 2+ from 0 to 2.6 mM. Cells were treated with thapsigargin (1 µM) during 10 min prior to the reintroduction of a Ca 2+ -containing solution (not shown); ( D ) Average Δpeak [Ca 2+ ] i increase at different time points under the conditions shown in C . ( E ) Percentage remaining Δpeak [Ca 2+ ] i at 7 min under the different KO conditions compared with control (set to 100%). The difference in [Ca 2+ ] i levels was analysed by two-way repeated measure ANOVA with Dunnett's multiple comparisons test at each time point. *P
    Figure Legend Snippet: siRNA knockdown of Stim1 , Orai1 and Trpc1 and gene silencing effects on SOCE. ( A and B ) mRNA levels for Stim1 , Orai1 and Trpc1 upon knockdown (KO) of each gene separately as well as upon the simultaneous silencing of Stim1 and Orai1 ; ( C ) traces of average [Ca 2+ ] i in response to an elevation of extracellular Ca 2+ from 0 to 2.6 mM. Cells were treated with thapsigargin (1 µM) during 10 min prior to the reintroduction of a Ca 2+ -containing solution (not shown); ( D ) Average Δpeak [Ca 2+ ] i increase at different time points under the conditions shown in C . ( E ) Percentage remaining Δpeak [Ca 2+ ] i at 7 min under the different KO conditions compared with control (set to 100%). The difference in [Ca 2+ ] i levels was analysed by two-way repeated measure ANOVA with Dunnett's multiple comparisons test at each time point. *P

    Techniques Used:

    The presence of STIM1, ORAI1 and TRPC1 in 3T3-L1 adipocytes. ( A ) mRNA levels of Stim1 , Orai1 and Trpc1 . Gene-specific mRNA levels are normalized against its respective β-actin mRNA level; ( B ) representative confocal images of adipocytes immunostained for TRPC1, ORAI1 and STIM1; ( C – E ) quantification of fluorescence intensity in adipocytes stained for ( C ) TRPC1, ( D ) ORAI1 and ( E ) STIM1 in five confocal images. The intensity is presented with the fluorescence intensity of Caveolin1 immunostaining in the same samples to show the intensity peak of the proteins of interest in relation to the plasma membrane (Caveolin1). * P
    Figure Legend Snippet: The presence of STIM1, ORAI1 and TRPC1 in 3T3-L1 adipocytes. ( A ) mRNA levels of Stim1 , Orai1 and Trpc1 . Gene-specific mRNA levels are normalized against its respective β-actin mRNA level; ( B ) representative confocal images of adipocytes immunostained for TRPC1, ORAI1 and STIM1; ( C – E ) quantification of fluorescence intensity in adipocytes stained for ( C ) TRPC1, ( D ) ORAI1 and ( E ) STIM1 in five confocal images. The intensity is presented with the fluorescence intensity of Caveolin1 immunostaining in the same samples to show the intensity peak of the proteins of interest in relation to the plasma membrane (Caveolin1). * P

    Techniques Used: Fluorescence, Staining, Immunostaining

    2) 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

    3) 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

    4) 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

    5) Product Images from "A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells"

    Article Title: A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.581678

    Effects of ORAI1 and TRPC1 knockdown on I CRAC in normal (NCM460) cells. A, representative I-V relationships ( left ) and current kinetics of I CRAC at −80 mV ( right ) in normal NCM460 cells transfected with scramble siRNA ( n = 12). B, representative
    Figure Legend Snippet: Effects of ORAI1 and TRPC1 knockdown on I CRAC in normal (NCM460) cells. A, representative I-V relationships ( left ) and current kinetics of I CRAC at −80 mV ( right ) in normal NCM460 cells transfected with scramble siRNA ( n = 12). B, representative

    Techniques Used: Transfection

    Effects of ORAI1 and TRPC1 knockdown on SOCE and SOCs in colon carcinoma (HT29) cells. A, ORAI1 knockdown inhibits SOCE. Real time PCR of HT29 cells transfected with siRNAs scramble and orai1 . Data were normalized to β-actin (mean ± S.E.,
    Figure Legend Snippet: Effects of ORAI1 and TRPC1 knockdown on SOCE and SOCs in colon carcinoma (HT29) cells. A, ORAI1 knockdown inhibits SOCE. Real time PCR of HT29 cells transfected with siRNAs scramble and orai1 . Data were normalized to β-actin (mean ± S.E.,

    Techniques Used: Real-time Polymerase Chain Reaction, Transfection

    Protein expression levels of SOCE-related channels and Stim proteins in normal (NCM460) and colon carcinoma (HT29) cells. A, Western blot assay of ORAI1 protein expression in normal and tumor cells. In this and the following panels, bars are mean ±
    Figure Legend Snippet: Protein expression levels of SOCE-related channels and Stim proteins in normal (NCM460) and colon carcinoma (HT29) cells. A, Western blot assay of ORAI1 protein expression in normal and tumor cells. In this and the following panels, bars are mean ±

    Techniques Used: Expressing, Western Blot

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    Alomone Labs orai1
    siRNA knockdown of Stim1 , <t>Orai1</t> and Trpc1 and gene silencing effects on SOCE. ( A and B ) mRNA levels for Stim1 , Orai1 and Trpc1 upon knockdown (KO) of each gene separately as well as upon the simultaneous silencing of Stim1 and Orai1 ; ( C ) traces of average [Ca 2+ ] i in response to an elevation of extracellular Ca 2+ from 0 to 2.6 mM. Cells were treated with thapsigargin (1 µM) during 10 min prior to the reintroduction of a Ca 2+ -containing solution (not shown); ( D ) Average Δpeak [Ca 2+ ] i increase at different time points under the conditions shown in C . ( E ) Percentage remaining Δpeak [Ca 2+ ] i at 7 min under the different KO conditions compared with control (set to 100%). The difference in [Ca 2+ ] i levels was analysed by two-way repeated measure ANOVA with Dunnett's multiple comparisons test at each time point. *P
    Orai1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    siRNA knockdown of Stim1 , Orai1 and Trpc1 and gene silencing effects on SOCE. ( A and B ) mRNA levels for Stim1 , Orai1 and Trpc1 upon knockdown (KO) of each gene separately as well as upon the simultaneous silencing of Stim1 and Orai1 ; ( C ) traces of average [Ca 2+ ] i in response to an elevation of extracellular Ca 2+ from 0 to 2.6 mM. Cells were treated with thapsigargin (1 µM) during 10 min prior to the reintroduction of a Ca 2+ -containing solution (not shown); ( D ) Average Δpeak [Ca 2+ ] i increase at different time points under the conditions shown in C . ( E ) Percentage remaining Δpeak [Ca 2+ ] i at 7 min under the different KO conditions compared with control (set to 100%). The difference in [Ca 2+ ] i levels was analysed by two-way repeated measure ANOVA with Dunnett's multiple comparisons test at each time point. *P

    Journal: Biochemical Journal

    Article Title: Extracellular ATP activates store-operated Ca2+ entry in white adipocytes: functional evidence for STIM1 and ORAI1

    doi: 10.1042/BCJ20170484

    Figure Lengend Snippet: siRNA knockdown of Stim1 , Orai1 and Trpc1 and gene silencing effects on SOCE. ( A and B ) mRNA levels for Stim1 , Orai1 and Trpc1 upon knockdown (KO) of each gene separately as well as upon the simultaneous silencing of Stim1 and Orai1 ; ( C ) traces of average [Ca 2+ ] i in response to an elevation of extracellular Ca 2+ from 0 to 2.6 mM. Cells were treated with thapsigargin (1 µM) during 10 min prior to the reintroduction of a Ca 2+ -containing solution (not shown); ( D ) Average Δpeak [Ca 2+ ] i increase at different time points under the conditions shown in C . ( E ) Percentage remaining Δpeak [Ca 2+ ] i at 7 min under the different KO conditions compared with control (set to 100%). The difference in [Ca 2+ ] i levels was analysed by two-way repeated measure ANOVA with Dunnett's multiple comparisons test at each time point. *P

    Article Snippet: Primary antibodies made in the rabbit (Alomone Labs, Jerusalem, Israel) directed against ORAI1 (1 : 1000), STIM1 (1 : 500) or TRPC1 (1 : 100) were incubated over night at 4°C in blocking solution.

    Techniques:

    The presence of STIM1, ORAI1 and TRPC1 in 3T3-L1 adipocytes. ( A ) mRNA levels of Stim1 , Orai1 and Trpc1 . Gene-specific mRNA levels are normalized against its respective β-actin mRNA level; ( B ) representative confocal images of adipocytes immunostained for TRPC1, ORAI1 and STIM1; ( C – E ) quantification of fluorescence intensity in adipocytes stained for ( C ) TRPC1, ( D ) ORAI1 and ( E ) STIM1 in five confocal images. The intensity is presented with the fluorescence intensity of Caveolin1 immunostaining in the same samples to show the intensity peak of the proteins of interest in relation to the plasma membrane (Caveolin1). * P

    Journal: Biochemical Journal

    Article Title: Extracellular ATP activates store-operated Ca2+ entry in white adipocytes: functional evidence for STIM1 and ORAI1

    doi: 10.1042/BCJ20170484

    Figure Lengend Snippet: The presence of STIM1, ORAI1 and TRPC1 in 3T3-L1 adipocytes. ( A ) mRNA levels of Stim1 , Orai1 and Trpc1 . Gene-specific mRNA levels are normalized against its respective β-actin mRNA level; ( B ) representative confocal images of adipocytes immunostained for TRPC1, ORAI1 and STIM1; ( C – E ) quantification of fluorescence intensity in adipocytes stained for ( C ) TRPC1, ( D ) ORAI1 and ( E ) STIM1 in five confocal images. The intensity is presented with the fluorescence intensity of Caveolin1 immunostaining in the same samples to show the intensity peak of the proteins of interest in relation to the plasma membrane (Caveolin1). * P

    Article Snippet: Primary antibodies made in the rabbit (Alomone Labs, Jerusalem, Israel) directed against ORAI1 (1 : 1000), STIM1 (1 : 500) or TRPC1 (1 : 100) were incubated over night at 4°C in blocking solution.

    Techniques: Fluorescence, Staining, Immunostaining