orai1  (Alomone Labs)


Bioz Verified Symbol Alomone Labs is a verified supplier
Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Alomone Labs orai1
    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/orai1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    orai1 - by Bioz Stars, 2022-08
    94/100 stars

    Images

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Alomone Labs mouse monoclonal anti orai1 antibody
    Hypoxia increases protein level (determined by immunofluorescence intensity) of STIM2 and <t>Orai1</t> only in PASMC, but not in CASMC. A and B : representative images ( A ) and summarized data ( B , means ± SE) showing STIM2 (green) and Orai1 (red) level in CASMC ( top ) and PASMC ( bottom ) under normoxic (Nor) and hypoxic (Hyp) conditions. The cells were dual-labeled with the anti-STIM2 antibody (green) and Orai1 antibody (red). Nuclei were stained with Hoechst (blue). Experiments were performed in 4 separate immunostaining procedure. ** P
    Mouse Monoclonal Anti Orai1 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/mouse monoclonal anti orai1 antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti orai1 antibody - by Bioz Stars, 2022-08
    94/100 stars
      Buy from Supplier

    93
    Alomone Labs mouse anti human anti kca3 1 biotinylated antibodies
    αPD-L1 treatment increases K + channel activity in HNSCC patients. (A) <t>KCa3.1</t> and (B) Kv1.3 conductance values (G) measured with and without the αPD-L1 antibody, atezolizumab (1 and 10 μg/ml for 6 h) in activated CD8 + PBTs of HNSCC patients. (C) KCa3.1 and (D) Kv1.3 G measured in presence of PD-L1 and αPD-1 antibody pembrolizumab in activated CD8 + PBTs of HDs. Activated cells were treated with plate-bound PD-L1 (PD-L1-Fc 10 μg/ml) +/- αPD-1 (untreated cells were used as a control) and activated for 72 h using PMA/Ionomycin. αPD-1 was added to treatment group for 6 h. Data in the lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal lines. The lower and upper error bars represents 10 th and 90 th percentile respectively, n = 8–23 cells from 3 HNSCC patients, n = 30 cells from 6 HDs (control and PD-L1) and n = 15 cells from 3 HDs (PD-L1 + αPD-1). Five cells were recorded for each individual donor. Data in (A,C,D) were analyzed by ANOVA on ranks test ( p
    Mouse Anti Human Anti Kca3 1 Biotinylated Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human anti kca3 1 biotinylated antibodies/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti human anti kca3 1 biotinylated antibodies - by Bioz Stars, 2022-08
    93/100 stars
      Buy from Supplier

    Image Search Results


    Hypoxia increases protein level (determined by immunofluorescence intensity) of STIM2 and Orai1 only in PASMC, but not in CASMC. A and B : representative images ( A ) and summarized data ( B , means ± SE) showing STIM2 (green) and Orai1 (red) level in CASMC ( top ) and PASMC ( bottom ) under normoxic (Nor) and hypoxic (Hyp) conditions. The cells were dual-labeled with the anti-STIM2 antibody (green) and Orai1 antibody (red). Nuclei were stained with Hoechst (blue). Experiments were performed in 4 separate immunostaining procedure. ** P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Hypoxia selectively upregulates cation channels and increases cytosolic [Ca2+] in pulmonary, but not coronary, arterial smooth muscle cells

    doi: 10.1152/ajpcell.00272.2017

    Figure Lengend Snippet: Hypoxia increases protein level (determined by immunofluorescence intensity) of STIM2 and Orai1 only in PASMC, but not in CASMC. A and B : representative images ( A ) and summarized data ( B , means ± SE) showing STIM2 (green) and Orai1 (red) level in CASMC ( top ) and PASMC ( bottom ) under normoxic (Nor) and hypoxic (Hyp) conditions. The cells were dual-labeled with the anti-STIM2 antibody (green) and Orai1 antibody (red). Nuclei were stained with Hoechst (blue). Experiments were performed in 4 separate immunostaining procedure. ** P

    Article Snippet: Cells were incubated overnight at 4°C with a mouse monoclonal anti-Orai1 antibody (1:100, catalog no. ALM-025, Alomone) ( ) and rabbit polyclonal anti-STIM2 antibody (1:200; catalog no. ab59342, Abcam, Cambridge, MA) ( ).

    Techniques: Immunofluorescence, Labeling, Staining, Immunostaining

    Comparison of basal protein expression levels of store-operated Ca 2+ channels (SOCC) (STIM1/2, Orai1/2) and ROCC (TRPC6) and hypoxia-induced changes of protein expression of SOCC (STIM1/2, Orai1/2) and ROCC (TRPC6) in CASMC and PASMC. A and B : representative images ( A ) and summarized data ( B , means ± SE) showing Western blot analysis of STIM1, STIM2, Orai1, Orai2, and TRPC6 in CASMC and PASMC ( n = 5 separate experiments). β-Actin was used as a control. * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Hypoxia selectively upregulates cation channels and increases cytosolic [Ca2+] in pulmonary, but not coronary, arterial smooth muscle cells

    doi: 10.1152/ajpcell.00272.2017

    Figure Lengend Snippet: Comparison of basal protein expression levels of store-operated Ca 2+ channels (SOCC) (STIM1/2, Orai1/2) and ROCC (TRPC6) and hypoxia-induced changes of protein expression of SOCC (STIM1/2, Orai1/2) and ROCC (TRPC6) in CASMC and PASMC. A and B : representative images ( A ) and summarized data ( B , means ± SE) showing Western blot analysis of STIM1, STIM2, Orai1, Orai2, and TRPC6 in CASMC and PASMC ( n = 5 separate experiments). β-Actin was used as a control. * P

    Article Snippet: Cells were incubated overnight at 4°C with a mouse monoclonal anti-Orai1 antibody (1:100, catalog no. ALM-025, Alomone) ( ) and rabbit polyclonal anti-STIM2 antibody (1:200; catalog no. ab59342, Abcam, Cambridge, MA) ( ).

    Techniques: Expressing, Western Blot

    Role of P2Y 12 and P2Y 13 receptors in ADP-mediated augmentation of store release in TREM2 knockout (KO) microglia. ( A ) Representative green (GCaMP6f) and red (tdTomato) channel overlay images of wild type (WT) (top) and TREM2 KO (bottom) induced pluripotent stem cell (iPSC)-microglia before and peak Ca 2+ response after ADP addition in Ca 2+ -free buffer. Scale bar = 20 μm. ( B ) Average trace (left panel) showing Ca 2+ response to 100 nM ADP in Ca 2+ -free buffer. Quantification of peak signal (right panel, n = 46–75 cells, two experiments, Mann–Whitney test). ( C ) Comparison of peak cytosolic Ca 2+ in response to ADP (2.5 μM ADP) in 1 mM Ca 2+ or Ca 2+ -free buffer (n = 38–96 cells , ordinary one-way ANOVA with multiple comparisons). ( D ) Volcano plot of differentially expressed genes from bulk RNA-sequencing of WT and TREM2 KO iPSC-microglia (n = 4). Genes for IP3R, STIM1, and ORAI1 are highlighted. ( E ) RNA normalized read counts for IP 3 receptor type 2 (ITPR2), PMCA1 (ATP2B1), SERCA2 (ATP2A2), SERCA3 (ATP2A3), STIM1, and ORAI1 in WT and TREM2 KO iPSC-microglia. Isoforms expressed lower than 10 reads in any sample are not considered expressed and are not shown. Relative expression of P2Y 12 and P2Y 13 receptors is shown for comparison of the relative fold change between WT and TREM2 KO cells. ( F, G ) Peak Ca 2+ response in Ca 2+ -free buffer after treatment with 1 or 10 μM ADP in the presence of P2Y 12 receptor antagonist PSB 0739 ( F ) or P2Y 13 receptor antagonist MRS 2211 ( G ), respectively. Cells were pretreated with 10 μM of PSB 0739 or 10 μM MRS 2211 for 30 min before imaging. (72–128 cells, F ; 83–117 cells, G ; representative of three experiments, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns, nonsignificant, **p

    Journal: eLife

    Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

    doi: 10.7554/eLife.73021

    Figure Lengend Snippet: Role of P2Y 12 and P2Y 13 receptors in ADP-mediated augmentation of store release in TREM2 knockout (KO) microglia. ( A ) Representative green (GCaMP6f) and red (tdTomato) channel overlay images of wild type (WT) (top) and TREM2 KO (bottom) induced pluripotent stem cell (iPSC)-microglia before and peak Ca 2+ response after ADP addition in Ca 2+ -free buffer. Scale bar = 20 μm. ( B ) Average trace (left panel) showing Ca 2+ response to 100 nM ADP in Ca 2+ -free buffer. Quantification of peak signal (right panel, n = 46–75 cells, two experiments, Mann–Whitney test). ( C ) Comparison of peak cytosolic Ca 2+ in response to ADP (2.5 μM ADP) in 1 mM Ca 2+ or Ca 2+ -free buffer (n = 38–96 cells , ordinary one-way ANOVA with multiple comparisons). ( D ) Volcano plot of differentially expressed genes from bulk RNA-sequencing of WT and TREM2 KO iPSC-microglia (n = 4). Genes for IP3R, STIM1, and ORAI1 are highlighted. ( E ) RNA normalized read counts for IP 3 receptor type 2 (ITPR2), PMCA1 (ATP2B1), SERCA2 (ATP2A2), SERCA3 (ATP2A3), STIM1, and ORAI1 in WT and TREM2 KO iPSC-microglia. Isoforms expressed lower than 10 reads in any sample are not considered expressed and are not shown. Relative expression of P2Y 12 and P2Y 13 receptors is shown for comparison of the relative fold change between WT and TREM2 KO cells. ( F, G ) Peak Ca 2+ response in Ca 2+ -free buffer after treatment with 1 or 10 μM ADP in the presence of P2Y 12 receptor antagonist PSB 0739 ( F ) or P2Y 13 receptor antagonist MRS 2211 ( G ), respectively. Cells were pretreated with 10 μM of PSB 0739 or 10 μM MRS 2211 for 30 min before imaging. (72–128 cells, F ; 83–117 cells, G ; representative of three experiments, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns, nonsignificant, **p

    Article Snippet: In response to reviewer comments, we generated Orai1 CRISPR-knockout iPSC-microglia and further tested the Orai1 Ab (Alomone, Cat# ALM-025, Clone 3F11/D10/B9) for immunostaining in WT and Orai1 KO iPSC-microglial cell line; antibody staining was found to be nonspecific.

    Techniques: Knock-Out, MANN-WHITNEY, RNA Sequencing Assay, Expressing, Imaging

    Regulation of store-operated Ca 2+ entry (SOCE) in induced pluripotent stem cell (iPSC)-microglia. ( A ) Average trace showing SOCE triggered in TREM2 knockout (KO) microglia via emptying endoplasmic reticulum (ER) Ca 2+ stores with thapsigargin (TG, 2 μM) in Ca 2+ -free buffer followed by readdition of 1 mM Ca 2+ in the absence (control, green trace) or presence (red trace) of the Orai channel inhibitor Synta66. Cells were pretreated with Synta66 (10 μM) for 30 min before experiment. Bar graph summary of the rate of Ca 2+ influx after readdition of 1 mM Ca 2+ (80–126 cells, Mann–Whitney test). ( B ) SOCE evoked by ADP (2.5 μM) in TREM2 KO microglia (green trace) using a similar Ca 2+ addback protocol. Red trace shows the effect of Synta66 on ADP-evoked SOCE. Right panel summarizes the rate of ADP-triggered Ca 2+ influx after readdition of 1 mM Ca 2+ (n = 125–154 cells, two experiments, Mann–Whitney test). ( C, D ) Cytosolic Ca 2+ response to ADP in TREM2 KO iPSC-microglia pretreated with 2-APB (50 μM) or Gd 3+ (5 μM) to block SOCE. Average traces ( C ), baseline-subtracted initial peak Ca 2+ responses to ADP ( D , left panel), and baseline-subtracted Ca 2+ after 5 min of ADP addition ( D , right panel) are shown (n = 41–74 cells, ordinary one-way ANOVA with multiple comparisons). ( E, F ) Role of Orai1 in TG- and ADP-evoked SOCE in iPSC-microglia. ( E ) Comparison of TG-evoked SOCE in WT and Orai1 KO cell showing average traces (left panel) and summary of SOCE rate (right panel; n = 42–54 cells, 3–4 experiments, Mann–Whitney test). ( F ) ADP-evoked SOCE in WT and Orai1 KO showing average traces (left panel) and summary of SOCE rate (right panel; n = 42–53 cells, 3–4 experiments, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns, nonsignificant, ****p

    Journal: eLife

    Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

    doi: 10.7554/eLife.73021

    Figure Lengend Snippet: Regulation of store-operated Ca 2+ entry (SOCE) in induced pluripotent stem cell (iPSC)-microglia. ( A ) Average trace showing SOCE triggered in TREM2 knockout (KO) microglia via emptying endoplasmic reticulum (ER) Ca 2+ stores with thapsigargin (TG, 2 μM) in Ca 2+ -free buffer followed by readdition of 1 mM Ca 2+ in the absence (control, green trace) or presence (red trace) of the Orai channel inhibitor Synta66. Cells were pretreated with Synta66 (10 μM) for 30 min before experiment. Bar graph summary of the rate of Ca 2+ influx after readdition of 1 mM Ca 2+ (80–126 cells, Mann–Whitney test). ( B ) SOCE evoked by ADP (2.5 μM) in TREM2 KO microglia (green trace) using a similar Ca 2+ addback protocol. Red trace shows the effect of Synta66 on ADP-evoked SOCE. Right panel summarizes the rate of ADP-triggered Ca 2+ influx after readdition of 1 mM Ca 2+ (n = 125–154 cells, two experiments, Mann–Whitney test). ( C, D ) Cytosolic Ca 2+ response to ADP in TREM2 KO iPSC-microglia pretreated with 2-APB (50 μM) or Gd 3+ (5 μM) to block SOCE. Average traces ( C ), baseline-subtracted initial peak Ca 2+ responses to ADP ( D , left panel), and baseline-subtracted Ca 2+ after 5 min of ADP addition ( D , right panel) are shown (n = 41–74 cells, ordinary one-way ANOVA with multiple comparisons). ( E, F ) Role of Orai1 in TG- and ADP-evoked SOCE in iPSC-microglia. ( E ) Comparison of TG-evoked SOCE in WT and Orai1 KO cell showing average traces (left panel) and summary of SOCE rate (right panel; n = 42–54 cells, 3–4 experiments, Mann–Whitney test). ( F ) ADP-evoked SOCE in WT and Orai1 KO showing average traces (left panel) and summary of SOCE rate (right panel; n = 42–53 cells, 3–4 experiments, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns, nonsignificant, ****p

    Article Snippet: In response to reviewer comments, we generated Orai1 CRISPR-knockout iPSC-microglia and further tested the Orai1 Ab (Alomone, Cat# ALM-025, Clone 3F11/D10/B9) for immunostaining in WT and Orai1 KO iPSC-microglial cell line; antibody staining was found to be nonspecific.

    Techniques: Knock-Out, MANN-WHITNEY, Blocking Assay

    Orai1 is ubiquitinated and degraded in a Crbn-dependent manner. a – d The lysates of 293T cells transfected with the indicated plasmids were incubated with anti-FLAG antibody-conjugated agarose beads. Bead-bound proteins were detected with the indicated antibodies. Data are representative of three a , c , d or four b independent experiments. e BMDMs derived from the indicated mice were treated with MG132 (5 µM) for 6 h and then lysed. Proteins in the lysates were detected with the indicated antibodies (top) and the levels of Orai1 were quantified (bottom). n = 3 experiments, mean ± SEM. The numbers in the graph indicate p values. NS not significant (two-way ANOVA).

    Journal: Nature Communications

    Article Title: Crbn modulates calcium influx by regulating Orai1 during efferocytosis

    doi: 10.1038/s41467-020-19272-0

    Figure Lengend Snippet: Orai1 is ubiquitinated and degraded in a Crbn-dependent manner. a – d The lysates of 293T cells transfected with the indicated plasmids were incubated with anti-FLAG antibody-conjugated agarose beads. Bead-bound proteins were detected with the indicated antibodies. Data are representative of three a , c , d or four b independent experiments. e BMDMs derived from the indicated mice were treated with MG132 (5 µM) for 6 h and then lysed. Proteins in the lysates were detected with the indicated antibodies (top) and the levels of Orai1 were quantified (bottom). n = 3 experiments, mean ± SEM. The numbers in the graph indicate p values. NS not significant (two-way ANOVA).

    Article Snippet: Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.

    Techniques: Transfection, Incubation, Derivative Assay, Mouse Assay

    The interaction of Orai1 with Crbn is attenuated during efferocytosis. a LR73 cells transfected with the indicated plasmids were incubated with TAMRA-stained apoptotic cells for 2 h and analyzed by flow cytometry. n = 3 experiments, mean ± SEM. The numbers in the graph indicate p values. NS not significant (one-way ANOVA). b The experiment was performed as in a . n = 3 experiments, mean ± SEM. NS not significant (two-tailed unpaired Student t test). c LR73 cells transfected with the indicated plasmids were lysed, and the proteins in the lysates were detected by immunoblotting. d BMDMs were incubated with apoptotic cells for the indicated durations were lysed, and the levels of Orai1 were detected with an anti-Orai1 antibody. e LR73 cells transfected with the indicated plasmids were incubated with apoptotic thymocytes for 60 min, and Orai1 ubiquitination were measured. f LR73 cells were incubated with apoptotic thymocytes for the indicated durations and the levels of Crbn were measured. g LR73 cells transfected with the indicated plasmids were incubated with apoptotic thymocytes for the indicated durations, and association between Crbn and Orai1 was measured. h LR73 cells stimulated with TAMRA-stained apoptotic cells were stained with an anti-Crbn antibody and observed using confocal microscopy (left) and Crbn localization was quantified (right). Scale bar, 20 µm. n = 8 cells for control and n = 25 cells for adding apoptotic cells, mean ± SEM. NS not significant (two-tailed unpaired Student t test). i LR73 cells incubated with apoptotic cells were lysed and fractionized. Proteins in the fractions were detected with the indicated antibodies. j LR73 cells transfected with the indicated plasmids were incubated with apoptotic cells, and association between Orai1 and Stim1 was measured. k 293T cells were transfected with the indicated plasmids, and association between Crbn and Oria1 was measured in the presence or absence of Stim1. Data are representative of three c – g , I , k or five j independent experiments. .

    Journal: Nature Communications

    Article Title: Crbn modulates calcium influx by regulating Orai1 during efferocytosis

    doi: 10.1038/s41467-020-19272-0

    Figure Lengend Snippet: The interaction of Orai1 with Crbn is attenuated during efferocytosis. a LR73 cells transfected with the indicated plasmids were incubated with TAMRA-stained apoptotic cells for 2 h and analyzed by flow cytometry. n = 3 experiments, mean ± SEM. The numbers in the graph indicate p values. NS not significant (one-way ANOVA). b The experiment was performed as in a . n = 3 experiments, mean ± SEM. NS not significant (two-tailed unpaired Student t test). c LR73 cells transfected with the indicated plasmids were lysed, and the proteins in the lysates were detected by immunoblotting. d BMDMs were incubated with apoptotic cells for the indicated durations were lysed, and the levels of Orai1 were detected with an anti-Orai1 antibody. e LR73 cells transfected with the indicated plasmids were incubated with apoptotic thymocytes for 60 min, and Orai1 ubiquitination were measured. f LR73 cells were incubated with apoptotic thymocytes for the indicated durations and the levels of Crbn were measured. g LR73 cells transfected with the indicated plasmids were incubated with apoptotic thymocytes for the indicated durations, and association between Crbn and Orai1 was measured. h LR73 cells stimulated with TAMRA-stained apoptotic cells were stained with an anti-Crbn antibody and observed using confocal microscopy (left) and Crbn localization was quantified (right). Scale bar, 20 µm. n = 8 cells for control and n = 25 cells for adding apoptotic cells, mean ± SEM. NS not significant (two-tailed unpaired Student t test). i LR73 cells incubated with apoptotic cells were lysed and fractionized. Proteins in the fractions were detected with the indicated antibodies. j LR73 cells transfected with the indicated plasmids were incubated with apoptotic cells, and association between Orai1 and Stim1 was measured. k 293T cells were transfected with the indicated plasmids, and association between Crbn and Oria1 was measured in the presence or absence of Stim1. Data are representative of three c – g , I , k or five j independent experiments. .

    Article Snippet: Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.

    Techniques: Transfection, Incubation, Staining, Flow Cytometry, Two Tailed Test, Confocal Microscopy

    Crbn interacts with Orai1. a , b Phagocytes from the indicated mice a or Crbn deleted 293T cells by CRISPR/Cas9 b were lysed and proteins in the lysates were detected using immunoblotting. Data are representative of three independent experiments. c The transcript levels of Orai1 were analyzed by conventional PCR (bottom) or qRT-PCR (top) using cDNA synthesized from total mRNA extracted from the indicated BMDMs. n = 3 experiments, mean ± SEM. NS not significant (two-tailed unpaired Student t test). d LR73 cells were transfected with the indicated plasmid. At 1 day after transfection, the cells were lysed and proteins in the lysates were detected with the indicated antibodies. Data are representative of three independent experiments. e , f 293T cells transfected with the indicated plasmids e or BMDMs f were lysed and then the lysates were incubated with anti-FLAG antibody-conjugated agarose beads e or an anti-Orai1 antibody and protein A/G agarose beads f . Bead-bound proteins were detected with the indicated antibodies. Data are representative of five e or three f independent experiments. IP immunoprecipitation, TCL total cell lysates. g – i 293T cells were transfected with the indicated plasmids. At 2 days after transfection, the cells were lysed and the lysates were incubated with agarose beads conjugated with glutathione g , i or an anti-FLAG antibody h . Bead-bound proteins were detected with the indicated antibodies. Data are representative of four g or three h , i independent experiments. j Yeast cells transformed with the indicated plasmids were plated on selective or non-selective media. Cells on the non-selective media indicate the number of cells plated. BD binding domain, AD activation domain.

    Journal: Nature Communications

    Article Title: Crbn modulates calcium influx by regulating Orai1 during efferocytosis

    doi: 10.1038/s41467-020-19272-0

    Figure Lengend Snippet: Crbn interacts with Orai1. a , b Phagocytes from the indicated mice a or Crbn deleted 293T cells by CRISPR/Cas9 b were lysed and proteins in the lysates were detected using immunoblotting. Data are representative of three independent experiments. c The transcript levels of Orai1 were analyzed by conventional PCR (bottom) or qRT-PCR (top) using cDNA synthesized from total mRNA extracted from the indicated BMDMs. n = 3 experiments, mean ± SEM. NS not significant (two-tailed unpaired Student t test). d LR73 cells were transfected with the indicated plasmid. At 1 day after transfection, the cells were lysed and proteins in the lysates were detected with the indicated antibodies. Data are representative of three independent experiments. e , f 293T cells transfected with the indicated plasmids e or BMDMs f were lysed and then the lysates were incubated with anti-FLAG antibody-conjugated agarose beads e or an anti-Orai1 antibody and protein A/G agarose beads f . Bead-bound proteins were detected with the indicated antibodies. Data are representative of five e or three f independent experiments. IP immunoprecipitation, TCL total cell lysates. g – i 293T cells were transfected with the indicated plasmids. At 2 days after transfection, the cells were lysed and the lysates were incubated with agarose beads conjugated with glutathione g , i or an anti-FLAG antibody h . Bead-bound proteins were detected with the indicated antibodies. Data are representative of four g or three h , i independent experiments. j Yeast cells transformed with the indicated plasmids were plated on selective or non-selective media. Cells on the non-selective media indicate the number of cells plated. BD binding domain, AD activation domain.

    Article Snippet: Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.

    Techniques: Mouse Assay, CRISPR, Polymerase Chain Reaction, Quantitative RT-PCR, Synthesized, Two Tailed Test, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Transformation Assay, Binding Assay, Activation Assay

    αPD-L1 treatment increases K + channel activity in HNSCC patients. (A) KCa3.1 and (B) Kv1.3 conductance values (G) measured with and without the αPD-L1 antibody, atezolizumab (1 and 10 μg/ml for 6 h) in activated CD8 + PBTs of HNSCC patients. (C) KCa3.1 and (D) Kv1.3 G measured in presence of PD-L1 and αPD-1 antibody pembrolizumab in activated CD8 + PBTs of HDs. Activated cells were treated with plate-bound PD-L1 (PD-L1-Fc 10 μg/ml) +/- αPD-1 (untreated cells were used as a control) and activated for 72 h using PMA/Ionomycin. αPD-1 was added to treatment group for 6 h. Data in the lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal lines. The lower and upper error bars represents 10 th and 90 th percentile respectively, n = 8–23 cells from 3 HNSCC patients, n = 30 cells from 6 HDs (control and PD-L1) and n = 15 cells from 3 HDs (PD-L1 + αPD-1). Five cells were recorded for each individual donor. Data in (A,C,D) were analyzed by ANOVA on ranks test ( p

    Journal: Frontiers in Pharmacology

    Article Title: Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

    doi: 10.3389/fphar.2021.742862

    Figure Lengend Snippet: αPD-L1 treatment increases K + channel activity in HNSCC patients. (A) KCa3.1 and (B) Kv1.3 conductance values (G) measured with and without the αPD-L1 antibody, atezolizumab (1 and 10 μg/ml for 6 h) in activated CD8 + PBTs of HNSCC patients. (C) KCa3.1 and (D) Kv1.3 G measured in presence of PD-L1 and αPD-1 antibody pembrolizumab in activated CD8 + PBTs of HDs. Activated cells were treated with plate-bound PD-L1 (PD-L1-Fc 10 μg/ml) +/- αPD-1 (untreated cells were used as a control) and activated for 72 h using PMA/Ionomycin. αPD-1 was added to treatment group for 6 h. Data in the lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal lines. The lower and upper error bars represents 10 th and 90 th percentile respectively, n = 8–23 cells from 3 HNSCC patients, n = 30 cells from 6 HDs (control and PD-L1) and n = 15 cells from 3 HDs (PD-L1 + αPD-1). Five cells were recorded for each individual donor. Data in (A,C,D) were analyzed by ANOVA on ranks test ( p

    Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

    Techniques: Activity Assay

    Short-time treatment with PD-L1 decreases KCa3.1 activity in a calmodulin-independent manner. (A) Flow cytometry histogram and geometric mean fluorescence intensity (gMFI) values (B) for CaM expression in activated CD8 + PBTs from HD donors ( n = 3) in the absence and presence of PD-L1. (C) Representative recordings of KCa3.1 currents in activated CD8 + PBTs from HDs showing the effect of PD-L1 (PD-L1-Fc, 10 μg/ml) and CaM (50 µM). (D) Average normalized KCa3.1 conductance ( G , nS) measured in the absence and presence of PD-L1, with and without CaM. All conductance values are normalized to average conductance value obtained from control recordings. Cells were pre-incubated with plate-bound PD-L1 (PD-L1-Fc,10 μg/ml, for 72 h) activated using anti-CD3/CD28 antibodies and treated with or without CaM (50 µM), that was delivered intracellularly via patch pipette during recordings ( n = 15–18 cells per group from 3 HDs). The values in panel (B) are represented as bar graphs. Each symbol represent an individual HD. The values are represented as mean ± SEM. The values in panel (D) are represented as box and whisker plots. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. Data in panel (B) were analyzed by t -test and data in panel (D) were analyzed by ANOVA on ranks ( p = 0.008) with Dunn’s post hoc analysis.

    Journal: Frontiers in Pharmacology

    Article Title: Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

    doi: 10.3389/fphar.2021.742862

    Figure Lengend Snippet: Short-time treatment with PD-L1 decreases KCa3.1 activity in a calmodulin-independent manner. (A) Flow cytometry histogram and geometric mean fluorescence intensity (gMFI) values (B) for CaM expression in activated CD8 + PBTs from HD donors ( n = 3) in the absence and presence of PD-L1. (C) Representative recordings of KCa3.1 currents in activated CD8 + PBTs from HDs showing the effect of PD-L1 (PD-L1-Fc, 10 μg/ml) and CaM (50 µM). (D) Average normalized KCa3.1 conductance ( G , nS) measured in the absence and presence of PD-L1, with and without CaM. All conductance values are normalized to average conductance value obtained from control recordings. Cells were pre-incubated with plate-bound PD-L1 (PD-L1-Fc,10 μg/ml, for 72 h) activated using anti-CD3/CD28 antibodies and treated with or without CaM (50 µM), that was delivered intracellularly via patch pipette during recordings ( n = 15–18 cells per group from 3 HDs). The values in panel (B) are represented as bar graphs. Each symbol represent an individual HD. The values are represented as mean ± SEM. The values in panel (D) are represented as box and whisker plots. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. Data in panel (B) were analyzed by t -test and data in panel (D) were analyzed by ANOVA on ranks ( p = 0.008) with Dunn’s post hoc analysis.

    Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

    Techniques: Activity Assay, Flow Cytometry, Fluorescence, Chick Chorioallantoic Membrane Assay, Expressing, Incubation, Transferring, Whisker Assay

    Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p

    Journal: Frontiers in Pharmacology

    Article Title: Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

    doi: 10.3389/fphar.2021.742862

    Figure Lengend Snippet: Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p

    Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

    Techniques: Inhibition, Patch Clamp, Transferring, Fluorescence, Chick Chorioallantoic Membrane Assay, Flow Cytometry

    αPD-1 treatment increases K + channel activity in HNSCC T cells. (A) Representative current traces of KCa3.1 and Kv1.3 channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs cells from a HNSCC patient in absence or presence of αPD-1 (10 μg/ml, for 6 h). Data are normalized to maximum current at +50 mV recorded using a ramp pulse protocol from −120 mV to +50 mV for 200 ms every 15 s. The holding potential used was −70 mV. (B,C) KCa3.1 (B) and Kv1.3 (C) conductance (G) measured in the absence or presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 68 cells without pembrolizumab and n = 55 cells with pembrolizumab from 14 patients). (D) Representative current traces of divalent free current (DVF) through CRAC channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs from a HNSCC patient. Data were recorded using a ramp pulse protocol from −100 to +100 mV with at holding potential of +30 mV every 1.5 s. Cells were perfused with 0 mM Ca 2+ solution (1 min) followed by 20 mM Ca 2+ (1 min) and DVF solutions (2 min, see methods) to amplify currents during recordings. (E) Peak DVF current values measured in absence and presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 34 cells without αPD-1 and n = 31 cells with αPD-1 from 8 patients). The values in panels (B,C) and (E) are represented as box plots: the horizontal line indicates the median; the lower box is the 25 th percentile; the upper box is the 75 th percentile; and the whiskers represent the 10 th and 90 th percentiles. (F) Ion channel expression (KCa3.1, Kv1.3, Orai1 and STIM1) in HNSCC patient T cells after treatment with αPD-1 (10 μg/ml for 6 h). Effect of αPD-1 treatment is shown as ratio of mean fluorescence intensity (MFI, fold change) values of treatment versus control group. Data are represented as scatter plot where each symbol represents an individual patient ( n = 4–5). Horizontal line represents mean values for each group. Data in panels (B,C,E) were analyzed by Mann-Whitney rank sum test.

    Journal: Frontiers in Pharmacology

    Article Title: Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

    doi: 10.3389/fphar.2021.742862

    Figure Lengend Snippet: αPD-1 treatment increases K + channel activity in HNSCC T cells. (A) Representative current traces of KCa3.1 and Kv1.3 channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs cells from a HNSCC patient in absence or presence of αPD-1 (10 μg/ml, for 6 h). Data are normalized to maximum current at +50 mV recorded using a ramp pulse protocol from −120 mV to +50 mV for 200 ms every 15 s. The holding potential used was −70 mV. (B,C) KCa3.1 (B) and Kv1.3 (C) conductance (G) measured in the absence or presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 68 cells without pembrolizumab and n = 55 cells with pembrolizumab from 14 patients). (D) Representative current traces of divalent free current (DVF) through CRAC channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs from a HNSCC patient. Data were recorded using a ramp pulse protocol from −100 to +100 mV with at holding potential of +30 mV every 1.5 s. Cells were perfused with 0 mM Ca 2+ solution (1 min) followed by 20 mM Ca 2+ (1 min) and DVF solutions (2 min, see methods) to amplify currents during recordings. (E) Peak DVF current values measured in absence and presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 34 cells without αPD-1 and n = 31 cells with αPD-1 from 8 patients). The values in panels (B,C) and (E) are represented as box plots: the horizontal line indicates the median; the lower box is the 25 th percentile; the upper box is the 75 th percentile; and the whiskers represent the 10 th and 90 th percentiles. (F) Ion channel expression (KCa3.1, Kv1.3, Orai1 and STIM1) in HNSCC patient T cells after treatment with αPD-1 (10 μg/ml for 6 h). Effect of αPD-1 treatment is shown as ratio of mean fluorescence intensity (MFI, fold change) values of treatment versus control group. Data are represented as scatter plot where each symbol represents an individual patient ( n = 4–5). Horizontal line represents mean values for each group. Data in panels (B,C,E) were analyzed by Mann-Whitney rank sum test.

    Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

    Techniques: Activity Assay, Incubation, Expressing, Fluorescence, MANN-WHITNEY