anti human orai1 extracellular fitc antibody  (Alomone Labs)

Journal: Cell reports

Article Title: NKD2 mediates stimulation-dependent ORAI1 trafficking to augment Ca 2+ entry in T cells

doi: 10.1016/j.celrep.2021.109603

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human ORAI1 (flow cytometry) , Alomone Labs , Cat# ACC-060; ACC-060-F.

Techniques: FLAG-tag, Western Blot, Flow Cytometry, Recombinant, Staining, Plasmid Preparation, Software, Imaging, Microscopy

Journal: Cell reports

Article Title: NKD2 mediates stimulation-dependent ORAI1 trafficking to augment Ca 2+ entry in T cells

doi: 10.1016/j.celrep.2021.109603

Figure Lengend Snippet: (A) Top: representative confocal images of Jurkat T cells expressing GFP-ORAI1 and NKD2-mCherry (NKD2-mCh) under resting conditions (top two panels) or 10 min after dropping onto anti-CD3 Ab-coated coverslips. Images represent different depths within the cells, as indicated. Data are representative of similar observations from 10 (resting) and 16 (anti-CD3 Ab-stimulated) cells in two independent experiments. Bottom: scatterplot showing Pearson’s correlation coefficient for colocalization between ORAI1 and NKD2 under resting (13 cells) or anti-CD3 Ab-stimulated (13 cells) conditions from two independent experiments (left). Scatterplot showing percentage of Orai1 + NKD2 + vesicles among the Orai1 + vesicles under resting (16 cells) and anti-CD3 Ab-stimulated (16 cells) conditions from two independent experiments (right). (B) Representative confocal images of Jurkat T cells expressing GFP-ORAI1 and NKD2-mCh under resting conditions (top panels) or 30 min after stimulation with staphylococcal enterotoxin E (SEE)-pulsed Raji B cells loaded with CellTracer Blue CMAC (7-amino-4-chloromethylcoumarin) dye. Leftmost panel shows bright-field images overlaid with Raji B cells in blue in the bottom panel. Data are representative of similar observations from 10 (resting) and 14 (stimulated) cells in two independent experiments. Scale bar, 5 μm. (C) Representative TIRF images of a Jurkat T cell expressing GFP-ORAI1 and NKD2-mCh at early (30 s, top panels) and later (200 s, bottom panels) time points after dropping onto anti-CD3 Ab-coated coverslip. The line graph on the right shows normalized (mean ± SEM) fluorescence intensities from indicated cell numbers (N) depicting kinetics of accumulation of ORAI1 and NKD2 at the site of contact between the cell and the coverslip. Scale bar, 5 μm.

Article Snippet: Human ORAI1 (flow cytometry) , Alomone Labs , Cat# ACC-060; ACC-060-F.

Techniques: Expressing, Fluorescence

Journal: Cell reports

Article Title: NKD2 mediates stimulation-dependent ORAI1 trafficking to augment Ca 2+ entry in T cells

doi: 10.1016/j.celrep.2021.109603

Figure Lengend Snippet: (A) Representative histograms showing levels of total ORAI1 protein in control, NKD2 KO, and ORAI1 KO Jurkat T cells after permeabilization and intracellular staining with anti-ORAI1 Ab. The bar graph shows average (±SEM) from four independent experiments. (B) Representative histograms showing levels of newly inserted PM-resident ORAI1 protein in control and NKD2 KO Jurkat T cells after TCR stimulation. Cells were incubated with saturating amounts of fluorescein isothiocyanate (FITC)-conjugated anti-ORAI1 Ab (to mask all PM-resident ORAI1) before stimulation with anti-CD3 Ab for 20 min. Subsequently, the cells were stained with unlabeled anti-ORAI1 Ab and APC-labeled secondary antibody without permeabilization to detect newly integrated surface ORAI1 protein. Bar graph shows average (±SEM) from four independent experiments. (C) Schematic of GFP-ORAI1-EC-HA construct. GFP is fused to the N terminus of ORAI1, and an HA tag is inserted in the second extracellular loop between transmembrane segments TM3 and TM4 of ORAI1. (D) Representative flow plots showing frequencies of ORAI1 high population in control and NKD2 KO Jurkat T cells expressing GFP-ORAI1-EC-HA at indicated time points after stimulation with anti-CD3 Ab. Non-permeabilized cells were stained with anti-HA Abs to label PM-localized ORAI1 as described in (B). Gating strategy is described in . (E) Bar graph showing surface expression of GFP-ORAI1-EC-HA in control, NKD2 KO Jurkat T cells, and NKD2 KO cells reconstituted for expression of NKD2 (KO + NKD2) after TCR stimulation (as demonstrated in D). Data show means ± SEM from three independent experiments. (F) Representative traces showing averaged SOCE from control (20 cells) and NKD2 KO (KO sgRNA #1, 20 cells) Jurkat T cells expressing GFP-ORAI1-EC-HA after TCR stimulation using anti-CD3 Abs in the presence of external solution containing 2 mM Ca 2+ . Bar graphs show averaged baseline-subtracted SOCE (±SEM) at the peak and later time point (600 s, sustained) from four independent experiments. *p < 0.05; **p < 0.005; ***p < 0.0001. See also .

Article Snippet: Human ORAI1 (flow cytometry) , Alomone Labs , Cat# ACC-060; ACC-060-F.

Techniques: Staining, Incubation, Labeling, Construct, Expressing

Journal: Cell reports

Article Title: NKD2 mediates stimulation-dependent ORAI1 trafficking to augment Ca 2+ entry in T cells

doi: 10.1016/j.celrep.2021.109603

Figure Lengend Snippet: (A) Line graph showing the averaged (±SEM) kinetics of normalized fluorescence changes in 20 Jurkat cells expressing ORAI1-EC-pHluorin upon exposure to acidic solution (pH 5.5) or ammonium chloride (50 mM). Images below show the fluorescence intensity of a representative cell at the time points indicated by numbers in the graph above. (B) Line graph showing the averaged (±SEM) kinetics of normalized fluorescence changes in 15 Jurkat T cells expressing ORAI1-EC-pHluorin after treatment with TEV protease at the indicated time points. Images below show the fluorescence intensity of a representative cell at the time points indicated by numbers in the graph above. (C) Representative flow plots showing frequencies of pHluorin + populations in control and NKD2 KO Jurkat T cells expressing ORAI1-EC-pHluorin under resting conditions or 20 min after stimulation with anti-CD3 and cross-linking Abs. Cells were untreated (−TEV) or treated with TEV protease (+TEV, 15 min) before stimulation. Bar graph shows means ± SEM of pooled technical replicates from three independent experiments. (D) Representative TIRF images of control (top panels) and NKD2 KO (bottom panels) Jurkat T cells expressing ORAI1-EC-pHluorin at the indicated time points after dropping onto anti-CD3 Ab-coated coverslips. The line graph (middle) shows normalized (mean ± SEM) fluorescence intensities from indicated cell numbers (N) depicting the kinetics of insertion of ORAI1 + vesicles on the PM. The scatter graph on the right depicts the number of vesicles inserted in control or NKD2 KO Jurkat T cell expressing ORAI1-EC-pHluorin. Each symbol represents data from an independent cell. Scale bar, 5 μm. **p < 0.005; ***p < 0.0001.

Article Snippet: Human ORAI1 (flow cytometry) , Alomone Labs , Cat# ACC-060; ACC-060-F.

Techniques: Fluorescence, Expressing

Journal: Cell reports

Article Title: NKD2 mediates stimulation-dependent ORAI1 trafficking to augment Ca 2+ entry in T cells

doi: 10.1016/j.celrep.2021.109603

Figure Lengend Snippet: (A) Schematic showing the domain structure of NKD2. NKD2 contains a myristoylation motif at the second glycine residue (G2), putative PKC phosphorylation sites (S31, S230, and T257), a Ca 2+ -binding EF-hand (known to interact with Dishevelled), a TGF-α-binding proline-rich domain, and a histidine-rich C terminus. (B) Left: representative immunoblot showing NKD2 expression in lysates from HeLa cells; unstimulated Jurkat T cells; or those stimulated with PMA, ionomycin, or PMA + ionomycin (left). β-Actin, loading control. Right: representative immunoblot for detection of NKD2 in lysates from HEK293T cells expressing NKD2 together with empty vector (Vec), WT PKC-θ, a constitutive active mutant of PKC-θ (CA), or a dominant-negative mutant of PKC-θ (DN) (right). Asterisk (*) indicates a band corresponding to phosphorylated NKD2. Data are representative of two independent experiments. (C) Top: representative immunoblot showing the molecular weight shift of WT and indicated mutants of NKD2 in lysates of HEK293T cells co-expressing constitutive active PKC-θ (CA PKC-θ; top two panels). Asterisk (*) indicates a band corresponding to phosphorylated NKD2. Bottom: representative immunoblot for detection of phosphorylated NKD2. Lysates of HEK293T cells co-expressing WT or S31A mutant of FLAG-tagged NKD2 with CA PKC-θ were immunoprecipitated with FLAG resin and immunoblotted for detection of phosphorylated NKD2 using phospho-serine Abs. Bottom: NKD2 from FLAG immunoprecipitates as loading controls. Images are representative of two independent experiments. (D) Frequencies of ORAI1 high population in control and NKD2 KO Jurkat T cells expressing GFP-ORAI1-EC-HA together with mCh-tagged WT or indicated mutants of NKD2. At the indicated time points after stimulation with anti-CD3 Abs, cells were stained with anti-HA antibodies without permeabilization to label PM-localized ORAI1. Data are average ± SEM from three independent experiments. (E) Representative confocal images of Jurkat T cells expressing GFP-ORAI1 and NKD2 WT -mCh, NKD2 G2A -mCh (G2A), NKD2 132DFD>AFA -mCh (EFmut), and NKD2 S31A -mCh (S31A) under resting conditions (top panels) or 20 min after dropping on stimulatory anti-CD3 antibody-coated coverslips (bottom panels). The top panels show images from the center of the cell, whereas the bottom panels show images from the bottom of the cell, which is in contact with the coverslip. Images are representative of at least 10 cells in each condition. Scale bar, 2 μm. (F) Representative traces showing averaged SOCE from NKD2 KO Jurkat T cells expressing empty vector (40 cells), NKD2 WT -mCh (35 cells), NKD2 G2A -mCh (30 cells), NKD2 132DFD>AFA -mCh (30 cells), or NKD2 S31A -mCh (30 cells) after TCR stimulation using anti-CD3 Abs in the presence of external solution containing 2 mM Ca 2+ . Bar graphs (right) show averaged baseline-subtracted SOCE (±SEM) at the peak and later time point (600 s, sustained) from three independent experiments. *p < 0.05; **p < 0.005; ***p < 0.0001. See also and .

Article Snippet: Human ORAI1 (flow cytometry) , Alomone Labs , Cat# ACC-060; ACC-060-F.

Techniques: Binding Assay, Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Dominant Negative Mutation, Molecular Weight, Immunoprecipitation, Staining

Journal: Cell reports

Article Title: NKD2 mediates stimulation-dependent ORAI1 trafficking to augment Ca 2+ entry in T cells

doi: 10.1016/j.celrep.2021.109603

Figure Lengend Snippet: (A) Representative histograms showing levels of NKD2 protein in primary T cells transduced with lentiviral vectors encoding scrambled sgRNA (control), NKD2 sgRNA #1, and NKD2 sgRNA #3, together with those encoding Cas9. Cells were permeabilized and stained with anti-NKD2 Ab. The bar graph (right) shows average (±SEM) from four independent experiments. (B) Representative traces showing averaged SOCE from primary control (73 cells) and NKD2 KO (KO sgRNA #1, 82 cells; KO sgRNA #3, 71 cells) T cells after TCR stimulation using anti-CD3 Abs, followed by ionomycin treatment in the presence of external solution containing 2 mM Ca 2+ . Bar graph (right) shows averaged baseline-subtracted SOCE (±SEM) from three independent experiments. (C) Representative histograms showing levels of newly inserted PM-resident ORAI1 protein in primary control and NKD2 KO effector T cells after TCR stimulation. Cells were transduced as described above to induce deletion of NKD2 and stimulated and stained as described in for detection of newly inserted PM-resident ORAI1. Bar graph (right) shows average (±SEM) of pooled technical replicates from three independent donors. (D) Representative flow plots showing expression of IFN-γ, IL-2, and TNF in control, NKD2 KO (sgRNA #1), and NKD2 KO (sgRNA #3) cells after re-stimulation with anti-CD3 and anti-CD28 Abs. Bar graph shows means ± SEM of pooled technical replicates from two independent experiments. (E) Representative histograms showing levels of T-bet in control, NKD2 KO (sgRNA #1), and NKD2 KO (sgRNA #3) cells. Bar graph shows means ± SEM of pooled technical replicates from two independent experiments. *p < 0.05; **p < 0.005; ***p < 0.0001. See also .

Article Snippet: Human ORAI1 (flow cytometry) , Alomone Labs , Cat# ACC-060; ACC-060-F.

Techniques: Transduction, Staining, Expressing

Journal: Cell reports

Article Title: NKD2 mediates stimulation-dependent ORAI1 trafficking to augment Ca 2+ entry in T cells

doi: 10.1016/j.celrep.2021.109603

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human ORAI1 (flow cytometry) , Alomone Labs , Cat# ACC-060; ACC-060-F.

Techniques: FLAG-tag, Western Blot, Flow Cytometry, Recombinant, Staining, Plasmid Preparation, Software, Imaging, Microscopy