Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 and stromal-interacting molecule 1 (STIM1) expression in mouse pulmonary arterial smooth muscle cells (PASMCs). A: RT-PCR products from cultured mouse PASMCs amplified using primers for mouse Orai1 (269 bp), STIM1 (473 bp), and β-actin (498 bp). Three separate RT-PCR reactions were performed in the presence (+) and absence (−) of reverse transcriptase (RT). B: Orai1, STIM1, and GAPDH proteins were detected in cultured mouse PASMCs using Western blot analysis. Experiments were performed in 5 separate Western blot analyses.

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Amplification, Reverse Transcription, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: STIM1 is associated with Orai1 to mediate CCE in mouse PASMCs. A and B: STIM1, Orai1, and GAPDH proteins were detected in nontransfected mouse PASMCs and in PASMCs transfected with 200 nM scrambled siRNA (negative control). The expression of STIM1 but not Orai1 or GAPDH was reduced significantly in cells transfected with 200 nM STIM1 siRNA. The expressions of STIM1 and Orai1 but not GAPDH were reduced significantly in cells transfected with both 200 nM STIM1 siRNA and 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: siRNA knockdown of STIM1 reduced the CPA-induced transient and sustained increase in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. siRNA knockdown of STIM1 and Orai1 further reduced the CPA-induced transient but not sustained increase in fura-2 fluorescence ratio in the presence of nifedipine. D: bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in negative control cells (filled bars, n = 103), in STIM1 siRNA-transfected cells (shaded bars, n = 148), and in STIM1 siRNA and Orai1 siRNA-transfected cells (open bars, n = 70). **P < 0.01, compared with negative control cells (ANOVA); ++P < 0.01, compared with negative control cells and STIM1 siRNA-transfected cells (ANOVA). E: siRNA knockdown of STIM1 reduced the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μM CPA in the presence of 10 μM nifedipine. siRNA knockdown of STIM1 and Orai1 further reduced the increase in Mn2+ quench of fura-2 fluorescence caused by CPA in the presence of nifedipine. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in negative control cells (filled bar, n = 125), in STIM1 siRNA-transfected cells (shaded bar, n = 151), and in STIM1 siRNA and Orai1 siRNA-transfected cells (open bar, n = 137). **P < 0.01, compared with negative control cells (ANOVA); ++P < 0.01, compared with the negative control cells and STIM1 siRNA-transfected cells (ANOVA).

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Transfection, Negative Control, Expressing, Western Blot, Knockdown, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: knockdown of Orai1 reduced CCE in STIM1-overexpressing mouse PASMCs. A and B: STIM1, Orai1, and GAPDH proteins were detected in cells infected with adenovirus containing green fluorescent protein (Ad-GFP) transfected with 200 nM scrambled siRNA. The expression of STIM1 but not Orai1 or GAPDH increased markedly in cells infected with STIM1-GFP-adenovirus (Ad-GFP-STIM1) transfected with scrambled siRNA. The expression of Orai1 but not STIM1 or GAPDH was reduced significantly in STIM1-overexpressing cells transfected with 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: overexpression of STIM1 in scrambled siRNA-transfected cells caused an increase in CPA-induced transient and sustained rise in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. The increases in fluorescence ratio were reduced in Orai1 siRNA-transfected cells overexpressed with STIM1. D: bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in GFP-infected cells transfected with scrambled siRNA (filled bars, n = 33), in STIM1-overexpressing cells transfected with scrambled siRNA (shaded bars, n = 65), and in STIM1-overexpressing cells transfected with Orai1 siRNA (open bars, n = 50). *P < 0.05, compared with GFP-infected cells transfected with scrambled siRNA and STIM1-overexpressing cells transfected with Orai1 siRNA (ANOVA). E: overexpression of STIM1 in scrambled siRNA-transfected cells caused a CPA-induced increase in Mn2+ quench of fura-2 fluorescence in the presence of 10 μM nifedipine. The increases in Mn2+ quench of fura-2 fluorescence was reduced in Orai1 siRNA-transfected cells overexpressed with STIM1. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in GFP-infected cells transfected with scrambled siRNA (filled bars, n = 40), in STIM1-overexpressing cells transfected with scrambled siRNA (shaded bars, n = 95), and in STIM1-overexpressing cells transfected with Orai1 siRNA (open bars, n = 57). **P < 0.01, compared with GFP-infected cells transfected with scrambled siRNA and STIM1-overexpressing cells transfected with Orai1 siRNA (ANOVA).

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Knockdown, Infection, Transfection, Expressing, Western Blot, Over Expression, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 coimmunoprecipitates with STIM1 in mouse PASMCs. A, left: Orai1 was detected in cultured mouse PASMCs in the absence and presence of store-depletion. Right: bar graph showing expression levels of Orai1 measured relative to GAPDH in control cells (denoted as 1, filled bar) and in cells subjected to store-depletion (open bar). Data are means ± SE of 7 separate Western blot analyses. B, left: STIM1 was detected in cultured mouse PASMCs in the absence and presence of store-depletion. Right: bar graph showing expression levels of STIM1 measured relative to GAPDH in control cells (denoted as 1, filled bar) and in cells subjected to store-depletion (open bar). Data are means ± SE of 7 separate Western blot analyses. C: STIM1 coimmunoprecipitated Orai1 in cultured mouse PASMCs in the absence and presence of store-depletion. STIM1 was first immunoprecipitated (IP) with EXBIO STIM1 antibody (10 μg), and the blot was subsequently probed with BD Biosciences STIM1 antibody (WB, 1:100). The blot was then probed for coimmunoprecipitation (co-IP) of Orai1 expression using Orai1 antibody (WB, 1:100, ProSci). Experiments were performed in 3 separate co-IP procedures and Western blot analyses.

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Cell Culture, Expressing, Control, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Colocalization of Orai1 and STIM1 in mouse PASMCs. A–D: staining of mouse cultured PASMCs after exposure of live cells with normal bath physiological salt solution (PSS). A: omission of Orai1 and STIM1 antibody resulted in no Orai1 or STIM1 staining. B–D: three representative cells dual-labeled with anti-Orai1 antibody (green) and STIM1 antibody (red). Orai1 and STIM1 colocalization (yellow/orange) is shown in the merged images. E–H: staining of mouse cultured PASMCs after exposure of live cells with Ca2+-free PSS containing 10 μM CPA. E: omission of Orai1 and STIM1 antibody resulted in no Orai1 or STIM1 staining. F–H: three representative cells dual-labeled with anti-Orai1 antibody (green) and STIM1 antibody (red). Colocalization of Orai1 and STIM1 is more apparent (yellow/orange) after store-depletion as shown in the merged images. Nuclei were stained with DAPI (blue). Experiments were performed in 3 separate immunostaining procedure, each with duplicate coverslips. Scale bars, 20 μm.

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Staining, Cell Culture, Labeling, Immunostaining

Journal: Cell reports

Article Title: Altered GM1 catabolism affects NMDAR-mediated Ca 2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model

doi: 10.1016/j.celrep.2024.114117

Figure Lengend Snippet: (A) Immunoblot analysis of cell fractions from 6-month-old WT and β-Gal − / − mice. Markers for the ER (Calnexin), PM (N-cadherin), and ER-PM junctions (ORAI1, STIM1, STIM2, VAPA, and VAPB) were enriched in their respective fractions. Immunoblots using HRP-conjugated cholera toxin B subunit (CTX-B) show high GM1 levels in β-Gal − / − fractions. (B) Representative HPTLC plate showing GM1 levels in the ER, PM, and ER-PM junctions isolated from 6-month-old WT and β-Gal − / − mice. STD, standard. Note: to detect GM1 in WT samples, the sample volume loaded was 3× that of the β-Gal − / − samples. (C) Quantification of GM1 levels from HPTLC plates performed in (B). n = 8. Values are expressed as median ± quartiles. Statistical analysis was performed using the Student’s t test; *** p < 0.001, **** p < 0.0001. (D) Representative HPTLC plate showing GM1 levels in ER-PM junctions isolated from 1-, 3-, and 6-month-old WT and β-Gal − / − mice. To detect GM1 in WT samples, the sample volume loaded was 3× that of the β-Gal − / − samples. (E) Quantification of GM1 levels from HPTLC plates performed in (D). n = 4. Values are expressed as median ± quartiles. Statistical analysis was performed using the Student’s t test with Welch’s correction; ns, not significant; *** p < 0.001, **** p < 0.0001.

Article Snippet: Rabbit anti-STIM1 antibody , Cell Signaling , Cat #: 4916; RRID: AB_2271287.

Techniques: Western Blot, High Performance Thin Layer Chromatography, Isolation

Journal: Cell reports

Article Title: Altered GM1 catabolism affects NMDAR-mediated Ca 2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model

doi: 10.1016/j.celrep.2024.114117

Figure Lengend Snippet:

Article Snippet: Rabbit anti-STIM1 antibody , Cell Signaling , Cat #: 4916; RRID: AB_2271287.

Techniques: Virus, Recombinant, Isolation, Magnetic Beads, Plasmid Preparation, Software

Journal: Molecular medicine reports

Article Title: STIM1 silencing inhibits the migration and invasion of A549 cells.

doi: 10.3892/mmr.2017.7010

Figure Lengend Snippet: Figure 1. Representative images of stromal interaction molecule 1 (A) high‑expression and (B) low‑expression in lung cancer tissues. (A-a and B-a) Magnification, 10x20; (A-b and B-b) magnification, 10x40.

Article Snippet: STIM1 (sc‐68897), matrix metalloproteinase (MMP)2 (sc-10736), MMP9 (sc-10737), N-cadherin (sc-7939) and E-cadherin (sc-7870) antibodies were purchased from Santa Cruz Biotechnology, Inc. Snail1 (#3879S) and Vimentin (#5741S) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques:

Journal: Molecular medicine reports

Article Title: STIM1 silencing inhibits the migration and invasion of A549 cells.

doi: 10.3892/mmr.2017.7010

Figure Lengend Snippet: Figure 2. Effects of STIM1 silencing on the migration and invasion of A549 cells. (A) Knockdown of STIM1 expression was confirmed by western blot analysis. (B) Cell migration to close the scratched wounds was reduced in the A549‑shSTIM1 cells compared with the A549‑shcon cells. (C) Reduced inva sion through the Matrigel matrix was observed in the A549‑shSTIM1 cells compared with the A549‑shcon cells. *P<0.05. shcon, control short hairpin RNA; shSTIM1, STIM1 short hairpin RNA; STIM1, stromal interaction molecule 1.

Article Snippet: STIM1 (sc‐68897), matrix metalloproteinase (MMP)2 (sc-10736), MMP9 (sc-10737), N-cadherin (sc-7939) and E-cadherin (sc-7870) antibodies were purchased from Santa Cruz Biotechnology, Inc. Snail1 (#3879S) and Vimentin (#5741S) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Migration, Knockdown, Expressing, Western Blot, Control, shRNA

Journal: Molecular medicine reports

Article Title: STIM1 silencing inhibits the migration and invasion of A549 cells.

doi: 10.3892/mmr.2017.7010

Figure Lengend Snippet: Figure 3. Effects of STIM1 silencing on A549 cell metastasis in vivo. The number of metastatic lung nodules was significantly reduced in the A549‑shSTIM1 group compared with the control group. *P<0.05. shcon, control short hairpin RNA; shSTIM1, STIM1 short hairpin RNA; STIM1, stromal interaction molecule 1.

Article Snippet: STIM1 (sc‐68897), matrix metalloproteinase (MMP)2 (sc-10736), MMP9 (sc-10737), N-cadherin (sc-7939) and E-cadherin (sc-7870) antibodies were purchased from Santa Cruz Biotechnology, Inc. Snail1 (#3879S) and Vimentin (#5741S) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: In Vivo, Control, shRNA

Journal: Molecular medicine reports

Article Title: STIM1 silencing inhibits the migration and invasion of A549 cells.

doi: 10.3892/mmr.2017.7010

Figure Lengend Snippet: Figure 4. Effects of STIM1 silencing on the expression of several proteins. Western blot analysis indicated that STIM1 silencing decreased the protein expression levels of Snail1, MMP2, MMP9 and Vimentin, and increased the protein expression levels of E‑cadherin. MMP, matrix metalloproteinase; shcon, control short hairpin RNA; shSTIM1, STIM1 short hairpin RNA; STIM1, stromal interaction molecule 1.

Article Snippet: STIM1 (sc‐68897), matrix metalloproteinase (MMP)2 (sc-10736), MMP9 (sc-10737), N-cadherin (sc-7939) and E-cadherin (sc-7870) antibodies were purchased from Santa Cruz Biotechnology, Inc. Snail1 (#3879S) and Vimentin (#5741S) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control, shRNA

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Co-localization and co-immunoprecipitation of TRPP2 and STIM1 in transfected HEK293 cells. a Upper penal: GFP-tagged TRPP2 (TRPP2-GFP) co-localized with endoplasmic reticulum maker (ER-DsRed); Lower penal: TRPP2-GFP co-localized with mCherry-tagged STIM1 (mCherry-STIM1). b Representative images showing co-immunoprecipitation followed by immunoblots [left, immunoblot with anti-GFP; right, immunoblot with anti-mCherry]. GFP or mCherry antibody pulled down the proteins from TRPP2-GFP and mCherry-STIM1 co-expressing HEK293 cells. The experiment was repeated 4 times

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Immunoprecipitation, Transfection, Western Blot, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: FRET efficiency of TRPP2-STIM1 interaction in TRPP2 and STIM1 co-expressing HEK293 cells. a Representative images showing GFP, mCherry and FRET efficiency channels. The cells expressed TRPP2-mCherry and STIM1-GFP (upper), mCherry-TRPP2 and STIM1-GFP (middle) or GFP-mCherry (lower) proteins respectively. b Summarized data showing FRET efficiency in transfected HEK293 cells (four groups: mCherry and GFP, mCherry-GFP, TRPP2-mCherry and STIM1-GFP, mCherry-TRPP2 and STIM1-GFP). Values are shown as the mean ± SEM ( n = 9–20 cells). * P < 0.05 for GFP and mCherry co-expression vs. GFP-mCherry expression; # P < 0.05 for TRPP2-mCherry and STIM1-GFP co-expression vs. mCherry-TRPP2 and STIM1-GFP co-expression. c Schematic diagram of full-length TRPP2 and its truncated derivatives: TRPP2 without N1 (2-111aa, ∆N1) and TRPP2 without N2 (112-221aa, ∆N2). d Representative images of co-immunoprecipitation experiments in HEK293 cells co-expressed with STIM1 plus GFP-tagged full-length TRPP2 (WT) or ∆N1 or ∆N2. IP, GFP antibody; IB, anti-EGFP antibody. The GFP-only vector was used as a negative control. e Summarized data showing the relative binding strength of STIM1 with TRPP2 (WT), or ∆N1 or ∆N2. The optical density of ∆N1 or ∆N2 blot was normalized to that of WT blot (= 100%) and expressed as the relative binding strength. Values are shown as the mean ± SEM ( n = 3). * P < 0.05 for STIM1 and ∆N1 or ∆N2 co-expression vs. STIM1 and TRPP2 co-expression

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Expressing, Transfection, Immunoprecipitation, Plasmid Preparation, Negative Control, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Effect of TRPP2-STIM1 interaction on store-operated Ca 2+ entry (SOCE) in TRPP2 and STIM1 co-expressing HEK293 cells. a - d Summary of data showing changes in Ca 2+ release ( a , c ) and SOCE ( b , d ) in HEK293 cells transfected with TRPP2 siRNA, STIM1 siRNA, STIM1, TRPP2 and/or dominant negative TRPP2 (D511V), and treated by ATP (10 μmol/L), 2APB (100 μmol/L) + ATP (10 μmol/L) and thapsigargin (TG, 2.5 μmol/L) for 8 min in Ca 2+ -free solution. SOCE was evoked by extracellular Ca 2+ (1 mmol/L) application. Values are shown as the mean ± SEM ( n = 3–5). * P < 0.05 compared with scrambled siRNA in each treatment. e Summarized data showing ATP (10 μmol/L)-induced SOCE in HEK293 cells co-expressed with STIM1 and GFP-tagged full-length TRPP2 (GFP-TRPP2) or TRPP2 without N1 (GFP-TRPP2-∆N1, deletion of 2-111aa in TRPP2) or TRPP2 without N2 (GFP-TRPP2-∆N2, deletion of 112-221aa in TRPP2). Values are shown as mean ± SEM ( n = 5). * P < 0.05 for STIM1 and GFP-TRPP2 or GFP-TRPP2-∆N2 co-expression vs. STIM1 and GFP-TRPP2-∆N1 co-expression

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Expressing, Transfection, Dominant Negative Mutation

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Co-immunoprecipitation and in situ proximity ligation assay (PLA) of TRPP2 and STIM1 in mouse aortic smooth muscle cells. ( a , b ) Immunoblots showing that anti-TRPP2 and anti-STIM1 recognized TRPP2 and STIM1 proteins, and co-immunoprecipitation followed by immunoblots ( a , immunoblot with anti-TRPP2; b , immunoblot with anti-STIM1). Proteins from the mouse aortic smooth muscle cells were immunoprecipitated with indicated antibody (+) or no antibody (−). ( c ) PLA analysis was used to detect the interaction between TRPP2 and STIM1. Representative images were displayed in the presence of anti-STIM1 antibody alone ((a)-(c)), or in the presence of anti-TRPP2 and anti-STIM1 antibodies ((d)-(f)). ((c), (f)) were merged with bright view. Red doted fluorescence showing positive signal. Nuclei were marked by DAPI staining (blue color). Scale bar represents 5 μm. The experiment was repeated 4 times

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Immunoprecipitation, In Situ, Proximity Ligation Assay, Western Blot, Fluorescence, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Role of TRPP2 in store-operated Ca 2+ entry (SOCE) and STIM1 puncta formation in mouse aortic smooth muscle cells. a Representative traces showing ATP (10 μmol/L)-induced Ca 2+ release in Ca 2+ -free solution and SOCE in the mouse aortic smooth muscle cells transfected with TRPP2, STIM1, both TRPP2 and STIM1, or scrambled siRNAs. b Summary of data showing changes in intracellular Ca 2+ concentration increase in response to extracellular ATP (10 μmol/L) or thapsigargin (TG, 2.5 μmol/L) application in the mouse aortic smooth muscle cells treated with or without 2APB (100 μmol/L) for 10 min in Ca 2+ -free solution. c Summary of data showing changes in intracellular Ca 2+ concentration increase in response to extracellular Ca 2+ (1 mmol/L) application in the mouse aortic smooth muscle cells treated by ATP (10 μmol/L), 2APB (100 μmol/L) + ATP (10 μmol/L) and thapsigargin (TG, 2.5 μmol/L) for 10 min in Ca 2+ -free solution. Values are shown as mean ± SEM ( n = 3–6 experiments). * P < 0.05 for scrambled siRNA vs. TRPP2 or STIM1 or TRPP2 + STIM1 siRNA transfection in each treatment. d Representative images showing STIM1 puncta formation in the mouse aortic smooth muscle cells transfected with TRPP2 siRNA or scrambled siRNA and treated by ATP (10 μmol/L), 2APB (100 μmol/L) + ATP (10 μmol/L) and thapsigargin (TG, 2.5 μmol/L) for 10 min in Ca 2+ -free solution. Scale bar represents 10 μm. The experiment was repeated 4 times

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Transfection, Concentration Assay

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Role of TRPP2 and STIM1 in Ca 2+ release and store-operated Ca 2+ entry (SOCE)-induced mouse aorta contraction. a Representative traces showing phenylephrine (Phe, 10 μmol/L)-induced contraction in Ca 2+ -free solution and extracellular Ca 2+ (2.5 mmol/L) re-addition-induced contraction in mice aortae. b - e Summarized data showing Phe-induced contraction in Ca 2+ -free solution ( b , d ) and extracellular Ca 2+ re-addition ( c , e )-induced contractions in mice aortae, which were transfected with TRPP2 siRNA ( b , c ), SITM1 siRNA ( d , e ) or scrambled siRNA. f - i Summarized data showing endothelin 1 (ET-1, 100 nmom/L)-induced contraction in Ca 2+ -free solution ( f , h ) and extracellular Ca 2+ re-addition ( g , i )-induced contractions in mice aortae transfected with TRPP2 siRNA ( f , g ), SITM1 siRNA ( h , i ) or scrambled siRNA. Values are shown as mean ± SEM ( n = 3–4 mice). * P < 0.05 for scrambled siRNA vs. TRPP2 siRNA or STIM1 siRNA transfection. j Representative traces showing thapsigargin (TG, 2.5 μmol/L)-induced contraction in Ca 2+ -free solution and extracellular Ca 2+ (2.5 mmol/L) re-addition-induced contraction in mice aortae. k - n Summarized data showing TG-induced contraction in Ca 2+ -free solution ( k , m ) and extracellular Ca 2+ re-addition ( l , n )-induced contraction in mice aortae, which were transfected with TRPP2 siRNA ( j - l ), SITM1 siRNA ( m - n ) or scrambled siRNAs. Values are shown as the mean ± SEM ( n = 3–4 mice). * P < 0.05 for TRPP2 siRNA or STIM1 siRNA vs. scrambled siRNA

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Role of TRPP2 and STIM1 in agonist-induced mouse aorta contraction. Phenylephrine (10 μmol/L, a , b , e , f ) and endothelin 1 (100 nmol/L, c , d , g , h ) concentration-dependently induced the contraction of the mice aortae transfected with TRPP2 ( a - d ), STIM1 ( e - h ) or scrambled siRNA. b , d , f , h The mice aortae were pretreated by heparin (1 mg/ml) using a reversible permeabilization loading procedure. Values are shown as mean ± SEM ( n = 4–6 mice). * P < 0.05 for scrambled siRNA vs. TRPP2 or STIM1 siRNA transfection

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Concentration Assay, Transfection

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: Cells were then stained for rabbit anti-human STIM1 (Proteintech) primary antibodies followed by secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher).

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

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 < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.

Article Snippet: Cells were then stained for rabbit anti-human STIM1 (Proteintech) primary antibodies followed by secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher).

Techniques: Inhibition, Patch Clamp, Transferring, Control, Fluorescence, Flow Cytometry