Anti-Protein orai-3 ORAI3 Antibody

Western blot analysis of ORAI3 in A20 cell lysate with ORAI3 antibody at (A) 1, (B) 2 and (C) 4 μg/mL.

Immunocytochemistry of ORAI3 in A20 cells with ORAI3 antibody at 10 μg/mL.

Immunohistochemistry of ORAI3 in mouse spleen tissue with ORAI3 antibody at 2 μg/mL.

Orai3 and Stim1 silencing blocks TGF-β induced SNAI1 transcription. NMuMG ( A – D ) or MDA-MB-231 ( E – H ) cells were transfected with indicated siRNAs for 96 h using a final concentration of 100 picomoles of siRNA. The cells were treated with TGF-β for 2 h prior to RNA and protein isolation. RNA was converted to cDNA and RT-PCR performed to analyze both the gene knockdown efficiency for each gene, and Snai1 transcript levels. Western blots were performed against each protein to test efficiency of knockdown and normalized using GAPDH. All data are representative of at least 3 biological replicates (See for quantitation). Statistical analyses were performed with Graphpad Prism software. * = p -value ≤ 0.05, ** = p -value ≤ 0.01; *** = p -value ≤ 0.001.
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Orai3 silencing blocks 2APB dependent increase in SOCE. Calcium imaging was performed in control ( A ) and Orai3 knockdown ( B ) NMuMG cells. Analog plots of the fluorescence ratio (340/380) from an average of 40–60 cells are shown. ( C ) Quantification (mean ± SD) of fluorescence ratio (340/380). All data are representative of at least 3 biological replicates. Statistical analyses were performed with Graphpad Prism software. * = p -value ≤ 0.05.
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Orai3 silencing inhibits both cell migration and Snai1 transcription in response to TGF-β. ( A ) NMuMG cells were treated as indicated with TGF-β, TGF-β+2APB or DMSO, in the presence or absence of siOrai3 and RNA isolated. RNA was converted to cDNA and analyzed by real-time PCR using primers specific to mouse Snai1, Snai2 or Twist1, and normalized to Gapdh. Data represent the average of 3 individual biological replicates. ( B ) Proteins isolated from the same cells as in (A) were evaluated for SNAI1 expression by immunoblotting. Antibody to ACTIN was used a loading control, and the blots are representative of at least 3 independent biological replicates. Blots were quantitated using the LiCOR imaging software and are represented as SNAI1/ACTB signal, after normalizing to DMSO control. Error bars represent SEM and statistical analyses were performed Graphpad PRISM. * = p -value ≤ 0.05, ** = p -value ≤ 0.01 relative to control. ( C ) Confluent NMuMG cells in a 6-well plate were serum starved for 4 h prior to treatment, and TGF-β (for 8 h) and/or 2APB (for 24 h) were added to the wells prior to wounding using a sterile 200 ul tip. Three representative fields were marked and imaged immediately at time of (0 h) and a time period after (8 h) wounding as described in materials and methods. The images were captured using an Olympus IX71 microscope camera. All data are representative of at least 3 biological replicates. Statistical analyses were performed with Graphpad Prism software. * = p -value ≤ 0.05, ** = p -value ≤ 0.01; *** = p -value ≤ 0.001; **** = p -value ≤ 0.0001. ( D ) Model for ORAI3-mediated Snai1 upregulation. AKT (green oval) pathway can be activated by both calcium (black circles) and by TGF-β signaling. 2APB prevents SOCE via ORAI1 and ORAI2, while increasing calcium influx through ORAI3. Activation of AKT triggers increased binding of p65 at the Snai1 promoter, leading to increased recruitment of Pol II and hence transcription of Snai1 .
Index in PubMed under a CC BY license. PMID: 30034631