orai1  (Alomone Labs)


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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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    Average 94 stars, based on 1 article reviews
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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 "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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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 anti neuropilin2 nrp2 polyclonal antibody
    <t>Nrp2</t> expression in activated macrophages during joint inflammation A. Nrp2 expression on immune cells in CIA synovium. The hind paws of CIA-induced mice were digested and subjected to flow cytometric analysis. The representative histograms of Nrp2 staining and the cumulative data are shown. Data are expressed means ± SD. N = 3 from 3 independent experiments. B. The characteristics of Nrp2-positive macrophages. The representative histograms of CD86 and MHC class II expression on joint-infiltrating macrophages (left) and their cumulative data (right) are shown. N = 6 from 2 independent experiments. C. The expression of Nrp2 on BMMs. BMMs were cultured under indicated conditions, and Nrp2 expression was determined by flow cytometry. The representative histograms and the cumulative data are shown. N = 3 from 3 independent experiments. D. NRP2 expression in synovium-infiltrating cells in RA. The deposited single-cell RNA-seq data was re-analyzed, and several cell types were defined by UMAP. Expression of NRP2 is indicated with purple dots. The statistical analyses were performed using an unpaired t-test.
    Anti Neuropilin2 Nrp2 Polyclonal Antibody, 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
    https://www.bioz.com/result/anti neuropilin2 nrp2 polyclonal antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti neuropilin2 nrp2 polyclonal antibody - by Bioz Stars, 2022-07
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    94
    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: 94/100, 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 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti orai1 - by Bioz Stars, 2022-07
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    Nrp2 expression in activated macrophages during joint inflammation A. Nrp2 expression on immune cells in CIA synovium. The hind paws of CIA-induced mice were digested and subjected to flow cytometric analysis. The representative histograms of Nrp2 staining and the cumulative data are shown. Data are expressed means ± SD. N = 3 from 3 independent experiments. B. The characteristics of Nrp2-positive macrophages. The representative histograms of CD86 and MHC class II expression on joint-infiltrating macrophages (left) and their cumulative data (right) are shown. N = 6 from 2 independent experiments. C. The expression of Nrp2 on BMMs. BMMs were cultured under indicated conditions, and Nrp2 expression was determined by flow cytometry. The representative histograms and the cumulative data are shown. N = 3 from 3 independent experiments. D. NRP2 expression in synovium-infiltrating cells in RA. The deposited single-cell RNA-seq data was re-analyzed, and several cell types were defined by UMAP. Expression of NRP2 is indicated with purple dots. The statistical analyses were performed using an unpaired t-test.

    Journal: bioRxiv

    Article Title: Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium

    doi: 10.1101/2022.02.12.480222

    Figure Lengend Snippet: Nrp2 expression in activated macrophages during joint inflammation A. Nrp2 expression on immune cells in CIA synovium. The hind paws of CIA-induced mice were digested and subjected to flow cytometric analysis. The representative histograms of Nrp2 staining and the cumulative data are shown. Data are expressed means ± SD. N = 3 from 3 independent experiments. B. The characteristics of Nrp2-positive macrophages. The representative histograms of CD86 and MHC class II expression on joint-infiltrating macrophages (left) and their cumulative data (right) are shown. N = 6 from 2 independent experiments. C. The expression of Nrp2 on BMMs. BMMs were cultured under indicated conditions, and Nrp2 expression was determined by flow cytometry. The representative histograms and the cumulative data are shown. N = 3 from 3 independent experiments. D. NRP2 expression in synovium-infiltrating cells in RA. The deposited single-cell RNA-seq data was re-analyzed, and several cell types were defined by UMAP. Expression of NRP2 is indicated with purple dots. The statistical analyses were performed using an unpaired t-test.

    Article Snippet: Anti-Neuropilin2 (Nrp2) polyclonal antibody was from Alomone Labs.

    Techniques: Expressing, Mouse Assay, Staining, Cell Culture, Flow Cytometry, RNA Sequencing Assay

    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

    Variable efficacy of Runx2-cre in deleting flox/flox Orai1 in osteoblasts. It is often assumed that promoter-cre constructs uniformly delete the target in all cells of an organ. Recent findings suggest that for reasons that are not clear this is sometimes not the case (see Text), we tested this by antibody labeling of Orai1 in wild type and Orai1fl/fl-Runx2cre bone. A . Rabbit antibody detects Orai1 in osteoblasts (upper left panel). Absence of antibody eliminates labeling (lower left panel). All sections are from one wild type animal. Left and right panels are of the same section. Osteoblasts are shown independently with phalloidin rhodamine (right panels). Fields are 200 µm across. B . In the wild type animal, osteoblasts are shown with the antibody at high power (fields 200 microns wide) and in phase of the same field (right). In the lower panels, the same field of a conditional KO animal. Some of the conditional KO cells (Orai1fl/fl-Runx2cre) do not label (arrows, phase and antibody label, lower panels). C . Lower power fields, 350 µm, of Orai1 labeled osteoblasts (surrounding tissue fluorescence is an artifact) in wild type bone (top) and conditional KO bone (bottom). Osteoblasts in the wild type label strongly, including surface cells of the bone (arrows, top). Some, but not all, of the osteoblasts in the conditional KO label (arrows, bottom). D . Western blot for Orai1 in cells expressing or not expressing the protein (See Fig 5 ). Thirty-five µg loads of cell protein from the isolates indicated were run on denaturing SDS-PAGE and blotted. This blot using the very specific Alomone antibody shows a trace of Orai1 even in the osteoblast KO preparation (left lane) and reduced, but not absent, Orai1 in the MSC KO (upper panel). The beta actin re-blot (lower panel) confirms similar protein loads.

    Journal: bioRxiv

    Article Title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

    doi: 10.1101/2022.02.14.480443

    Figure Lengend Snippet: Variable efficacy of Runx2-cre in deleting flox/flox Orai1 in osteoblasts. It is often assumed that promoter-cre constructs uniformly delete the target in all cells of an organ. Recent findings suggest that for reasons that are not clear this is sometimes not the case (see Text), we tested this by antibody labeling of Orai1 in wild type and Orai1fl/fl-Runx2cre bone. A . Rabbit antibody detects Orai1 in osteoblasts (upper left panel). Absence of antibody eliminates labeling (lower left panel). All sections are from one wild type animal. Left and right panels are of the same section. Osteoblasts are shown independently with phalloidin rhodamine (right panels). Fields are 200 µm across. B . In the wild type animal, osteoblasts are shown with the antibody at high power (fields 200 microns wide) and in phase of the same field (right). In the lower panels, the same field of a conditional KO animal. Some of the conditional KO cells (Orai1fl/fl-Runx2cre) do not label (arrows, phase and antibody label, lower panels). C . Lower power fields, 350 µm, of Orai1 labeled osteoblasts (surrounding tissue fluorescence is an artifact) in wild type bone (top) and conditional KO bone (bottom). Osteoblasts in the wild type label strongly, including surface cells of the bone (arrows, top). Some, but not all, of the osteoblasts in the conditional KO label (arrows, bottom). D . Western blot for Orai1 in cells expressing or not expressing the protein (See Fig 5 ). Thirty-five µg loads of cell protein from the isolates indicated were run on denaturing SDS-PAGE and blotted. This blot using the very specific Alomone antibody shows a trace of Orai1 even in the osteoblast KO preparation (left lane) and reduced, but not absent, Orai1 in the MSC KO (upper panel). The beta actin re-blot (lower panel) confirms similar protein loads.

    Article Snippet: Orai1 labelingRabbit polyclonal-anti Orai1 was used for tissue labeling [ ]; rabbit anti-Orai1 (extracellular) antibody was from Alomone Labs (ACC-062, Jerusalem, Israel).

    Techniques: Construct, Antibody Labeling, Labeling, Fluorescence, Western Blot, Expressing, SDS Page

    Key features of WT and conditional KO animals on static and dynamic histomorphometry. A . Bone volume/total volume (BV/TV) of vertebrae. Vertebral bone is significantly reduced (N=7, p=0.01) in the Orai1fl/fl-Runx2cre (Orai1 cKO) mice compared to controls. The greater variability in the conditional-knockout bone is thought to reflect variable cre excision; this variability is also apparent in the cortical section thickness ( Fig 2 ; see text). B . Trabecular thickness is significantly decreased in the Orai1 conditional knockout mice (N=7, p

    Journal: bioRxiv

    Article Title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

    doi: 10.1101/2022.02.14.480443

    Figure Lengend Snippet: Key features of WT and conditional KO animals on static and dynamic histomorphometry. A . Bone volume/total volume (BV/TV) of vertebrae. Vertebral bone is significantly reduced (N=7, p=0.01) in the Orai1fl/fl-Runx2cre (Orai1 cKO) mice compared to controls. The greater variability in the conditional-knockout bone is thought to reflect variable cre excision; this variability is also apparent in the cortical section thickness ( Fig 2 ; see text). B . Trabecular thickness is significantly decreased in the Orai1 conditional knockout mice (N=7, p

    Article Snippet: Orai1 labelingRabbit polyclonal-anti Orai1 was used for tissue labeling [ ]; rabbit anti-Orai1 (extracellular) antibody was from Alomone Labs (ACC-062, Jerusalem, Israel).

    Techniques: Mouse Assay, Knock-Out

    Cross sections of vertebral cortex from wild-type (A) and Orai1 f/f -Runx2-cre conditional KO (B) animals. Images of H E stained histologic sections from upper lumbar vertebrae (top panels) are 1 mm wide; images from microCT scans of lower lumbar vertebrae (middle and lower panels) are 1.4 mm wide, with trabecular bone deleted. A . Wild type cortex with typical smooth bone showed relatively uniform thickness. B . The Orai1fl/fl-Runx2cre (abbreviated Orai1 cKO) animals had irregularly thinned regions (arrow) but no other distinguishing features. This is in keeping with the appearance of the bone surface in the three-dimensional reconstructions (see Fig 1). C . Cortical bone thickness, though variable, is reduced on average in Orai1fl/fl-Runx2cre (Orai1 cKO) animals (p = 0.009, N=8).

    Journal: bioRxiv

    Article Title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

    doi: 10.1101/2022.02.14.480443

    Figure Lengend Snippet: Cross sections of vertebral cortex from wild-type (A) and Orai1 f/f -Runx2-cre conditional KO (B) animals. Images of H E stained histologic sections from upper lumbar vertebrae (top panels) are 1 mm wide; images from microCT scans of lower lumbar vertebrae (middle and lower panels) are 1.4 mm wide, with trabecular bone deleted. A . Wild type cortex with typical smooth bone showed relatively uniform thickness. B . The Orai1fl/fl-Runx2cre (abbreviated Orai1 cKO) animals had irregularly thinned regions (arrow) but no other distinguishing features. This is in keeping with the appearance of the bone surface in the three-dimensional reconstructions (see Fig 1). C . Cortical bone thickness, though variable, is reduced on average in Orai1fl/fl-Runx2cre (Orai1 cKO) animals (p = 0.009, N=8).

    Article Snippet: Orai1 labelingRabbit polyclonal-anti Orai1 was used for tissue labeling [ ]; rabbit anti-Orai1 (extracellular) antibody was from Alomone Labs (ACC-062, Jerusalem, Israel).

    Techniques: Staining

    Surface of wild type and Orai1fl/fl-Runx2cre fourth lumbar vertebrae. Bruker CTvox software-generated three-dimensional images of vertebrae reconstructed from microCT scans at 5 µm resolution. All animals were homozygous for floxed Orai1; the conditional knockouts (lower panels) are Runx2-cre positive. A . Representative vertebrae from control animals. Apart from sites of blood vessel entry, the surface of the bone is smooth, typical for mice at four months of age. B . Representative vertebrae from Orai1fl/fl-Runx2cre animals. In contrast to the control vertebrae, the bone surface appears irregular with patchy darker areas representing regions of reduced bone.

    Journal: bioRxiv

    Article Title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

    doi: 10.1101/2022.02.14.480443

    Figure Lengend Snippet: Surface of wild type and Orai1fl/fl-Runx2cre fourth lumbar vertebrae. Bruker CTvox software-generated three-dimensional images of vertebrae reconstructed from microCT scans at 5 µm resolution. All animals were homozygous for floxed Orai1; the conditional knockouts (lower panels) are Runx2-cre positive. A . Representative vertebrae from control animals. Apart from sites of blood vessel entry, the surface of the bone is smooth, typical for mice at four months of age. B . Representative vertebrae from Orai1fl/fl-Runx2cre animals. In contrast to the control vertebrae, the bone surface appears irregular with patchy darker areas representing regions of reduced bone.

    Article Snippet: Orai1 labelingRabbit polyclonal-anti Orai1 was used for tissue labeling [ ]; rabbit anti-Orai1 (extracellular) antibody was from Alomone Labs (ACC-062, Jerusalem, Israel).

    Techniques: Software, Generated, Mouse Assay

    Elimination of Orai1 results in profoundly reduces OB differentiation and mineralization from OB precursors. Osteoblasts isolated as described in the methods section from control or Runx2-cre floxed (Orai1 f/f -Runx2-cre) conditional knock-out animals, were incubated 24 days in osteoblast mineralization medium and analyzed by histologic staining. Each well illustrated is 2 cm across. A . Von Kossa staining for mineral. Wild type cells made mineral nodules, but there were only rare and small nodules in Orai1 knockout cell cultures. Representative culture wells for control (WT) and conditional knockout (cKO) cells are shown on the left. Staining was quantified for four samples of each genotype. Mineralized matrix production appeared significantly reduced in cultures of Orai1-deficient osteoblasts (p

    Journal: bioRxiv

    Article Title: The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

    doi: 10.1101/2022.02.14.480443

    Figure Lengend Snippet: Elimination of Orai1 results in profoundly reduces OB differentiation and mineralization from OB precursors. Osteoblasts isolated as described in the methods section from control or Runx2-cre floxed (Orai1 f/f -Runx2-cre) conditional knock-out animals, were incubated 24 days in osteoblast mineralization medium and analyzed by histologic staining. Each well illustrated is 2 cm across. A . Von Kossa staining for mineral. Wild type cells made mineral nodules, but there were only rare and small nodules in Orai1 knockout cell cultures. Representative culture wells for control (WT) and conditional knockout (cKO) cells are shown on the left. Staining was quantified for four samples of each genotype. Mineralized matrix production appeared significantly reduced in cultures of Orai1-deficient osteoblasts (p

    Article Snippet: Orai1 labelingRabbit polyclonal-anti Orai1 was used for tissue labeling [ ]; rabbit anti-Orai1 (extracellular) antibody was from Alomone Labs (ACC-062, Jerusalem, Israel).

    Techniques: Isolation, Knock-Out, Incubation, Staining

    Schematic representation of the involvement of SERCA2a in the physiological control of SOCE GPCR - G-protein coupled receptor; PLC - phospholipase C; NFAT - nuclear factor of activated T lymphocytes; P - phosphate; IP 3 - inositol-1,4,5-trisphosphate, IP 3 R - IP 3 receptor; SR/ER sarco/endoplasmic reticulum; SERCA - SE/ER Ca 2+ ATPase; STIM1 - Stromal Interaction Molecule 1, ORAI1 - the pore forming unit.

    Journal: Journal of molecular and cellular cardiology

    Article Title: SERCA2a controls the mode of agonist-induced intracellular Ca2+ signal, transcription factor NFAT and proliferation in human vascular smooth muscle cells

    doi: 10.1016/j.yjmcc.2010.12.016

    Figure Lengend Snippet: Schematic representation of the involvement of SERCA2a in the physiological control of SOCE GPCR - G-protein coupled receptor; PLC - phospholipase C; NFAT - nuclear factor of activated T lymphocytes; P - phosphate; IP 3 - inositol-1,4,5-trisphosphate, IP 3 R - IP 3 receptor; SR/ER sarco/endoplasmic reticulum; SERCA - SE/ER Ca 2+ ATPase; STIM1 - Stromal Interaction Molecule 1, ORAI1 - the pore forming unit.

    Article Snippet: The following primary antibodies were used: IID8 (sc-53010, Santa Cruz Biotechnology), anti-SERCA2a and anti-SERCA2b [ ], anti-RyR2 [ ], anti- n on- m uscular myosin heavy chain B (NM-B) (Ab 684, Abcam), anti-smooth muscle m yosin h eavy c hain (MHC) (M3558, Dako Cytomation), anti-Cyclin D1 (556470, BD Biosciences), anti-PP2B (calcineurin, 556350, BD Biosciences), anti-STIM1 (ACC-63, Alomone labs), anti-Orai2 (ACC-061, Alomone labs), anti-ORAI1 (ACC-60, Alomone lab), anti-ORAI1 (sc-68895), anti-Cav1.2 calcium channel (L-type Ca2+ channel α1C subunit) (75053, NeuroMab); anti-h-calponin (C2687, Sigma-Aldrich), anti-caldesmon (C4562, Sigma-Aldrich); anti- g lycer a ldehyde 3- p hosphate d e h ydrogenase (GAPDH) (sc-47424, Santa Cruz Biotechnology).

    Techniques: Planar Chromatography

    SERCA2a prevents the formation of STIM-1/ORAI1 complex in cultured hCASMCs A. Effect of SERCA2a gene transfer on the expression of SOC sub-units. mRNA level quantified by real-time PCR was normalized to the value obtained in Ad-βGal-infected cells. Histograms show the means ± SEM of three experiments. B. Cells were infected for 4 days with Ad-βGal or Ad-S2a. Total protein extracts (50μg) were loaded. Left panel: western blot showing the expression of ORAI1, ORAI2 and STIM1 in whole-cell lysates. Right panel : histograms showing the relative ratio of ORAI1, ORAI2 and STIM1 normalized to GAPDH in three experiments. C. Whole-cell lysates were immunoprecipitated (IP) with an anti-STIM1 antibody, resolved on SDS/PAGE and immunobloted for ORAI1 or ORAI2. Membranes were reprobed for STIM1 for protein loading control. Histograms showing the mean (n=4) relative ratio of ORAI1 (left panel) and ORAI2 (right panel) normalized to STIM1 and arbitrary considered as 100% for Ad-βGal infected cells.

    Journal: Journal of molecular and cellular cardiology

    Article Title: SERCA2a controls the mode of agonist-induced intracellular Ca2+ signal, transcription factor NFAT and proliferation in human vascular smooth muscle cells

    doi: 10.1016/j.yjmcc.2010.12.016

    Figure Lengend Snippet: SERCA2a prevents the formation of STIM-1/ORAI1 complex in cultured hCASMCs A. Effect of SERCA2a gene transfer on the expression of SOC sub-units. mRNA level quantified by real-time PCR was normalized to the value obtained in Ad-βGal-infected cells. Histograms show the means ± SEM of three experiments. B. Cells were infected for 4 days with Ad-βGal or Ad-S2a. Total protein extracts (50μg) were loaded. Left panel: western blot showing the expression of ORAI1, ORAI2 and STIM1 in whole-cell lysates. Right panel : histograms showing the relative ratio of ORAI1, ORAI2 and STIM1 normalized to GAPDH in three experiments. C. Whole-cell lysates were immunoprecipitated (IP) with an anti-STIM1 antibody, resolved on SDS/PAGE and immunobloted for ORAI1 or ORAI2. Membranes were reprobed for STIM1 for protein loading control. Histograms showing the mean (n=4) relative ratio of ORAI1 (left panel) and ORAI2 (right panel) normalized to STIM1 and arbitrary considered as 100% for Ad-βGal infected cells.

    Article Snippet: The following primary antibodies were used: IID8 (sc-53010, Santa Cruz Biotechnology), anti-SERCA2a and anti-SERCA2b [ ], anti-RyR2 [ ], anti- n on- m uscular myosin heavy chain B (NM-B) (Ab 684, Abcam), anti-smooth muscle m yosin h eavy c hain (MHC) (M3558, Dako Cytomation), anti-Cyclin D1 (556470, BD Biosciences), anti-PP2B (calcineurin, 556350, BD Biosciences), anti-STIM1 (ACC-63, Alomone labs), anti-Orai2 (ACC-061, Alomone labs), anti-ORAI1 (ACC-60, Alomone lab), anti-ORAI1 (sc-68895), anti-Cav1.2 calcium channel (L-type Ca2+ channel α1C subunit) (75053, NeuroMab); anti-h-calponin (C2687, Sigma-Aldrich), anti-caldesmon (C4562, Sigma-Aldrich); anti- g lycer a ldehyde 3- p hosphate d e h ydrogenase (GAPDH) (sc-47424, Santa Cruz Biotechnology).

    Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Infection, Western Blot, Immunoprecipitation, SDS Page