Journal: The Eurasian journal of medicine

Article Title: Electron Microscopic and Immunohistochemical Examination of the Effect of 2-Aminoethoxydiphenyl Borate on Optic Nerve Injury in A Rat Model.

doi: 10.5152/eurasianjmed.2020.19089

Figure Lengend Snippet: Figure 3. a, b Light photomicrographs of Orai1 (a-a2) and STIM1 (b-b2) immunohistochemistry of optic nerve tissues. (a, b) Sham groups; (a1-b1) AIR groups; (a2-b2) AIR10 groups. (+), mild staining; (++), moderate staining; (+++), severe staining. Bars: 50 μm.

Article Snippet: The sections were then incubated at room temperature with drops of Orai1 (Santa Cruz; dilution 1/100) and STIM1 (Santa Cruz; dilution 1/100) antibodies (no primary antibody was added to the negative control sections) for 60 min.

Techniques: Immunohistochemistry, Staining

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: Journal of Innate Immunity

Article Title: Mannan-Binding Lectin Reduces Epithelial-Mesenchymal Transition in Pulmonary Fibrosis via Inactivating the Store-Operated Calcium Entry Machinery

doi: 10.1159/000524693

Figure Lengend Snippet: Primers for the target genes

Article Snippet: The membranes were then stained with HRP-conjugated secondary antibody (S0001; Affinity Biosciences) at room temperature for another 1 h. Antibodies involved in this subsection are listed below: E-cadherin (14472, mouse anti-human monoclonal antibody, 1:1,000 dilution; Cell Signaling Technology), N-cadherin (22018-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), α-SMA (14395-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), vimentin (10366-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), Orai1 (66223-1-Ig, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), Stim1 (5668, rabbit anti-human monoclonal antibody, 1:1,000 dilution; Cell Signaling Technology), SGK1 (23394-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), PDK1 (17086-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), GAPDH (60004-1-Ig, mouse anti-human monoclonal antibody, 1:1,000 dilution; Proteintech), and Ub (sc-8017, mouse anti-human monoclonal antibody, 1:1,000 dilution; Santa Cruz Biotechnology).

Techniques:

Journal: Journal of Innate Immunity

Article Title: Mannan-Binding Lectin Reduces Epithelial-Mesenchymal Transition in Pulmonary Fibrosis via Inactivating the Store-Operated Calcium Entry Machinery

doi: 10.1159/000524693

Figure Lengend Snippet: MBL mediated Orai1 ubiquitination. a, b HBE cells were incubated with 10-ng/mL TGF-β in the presence or absence of 10-μg/mL MBL for 24 h. a mRNA levels of Orai1 and Stim1 were assessed by quantitative RT-PCR. b Orai1 and Stim1 expression were determined by Western blot. c Orai1 and Stim1 levels in mice lung tissues were detected by Western blot. d HBE cells were incubated with 10-ng/mL TGF-β in the presence or absence of 10-μg/mL MBL for 24 h. Cells were treated with 10-μM MG132 4 h before being harvested. The ubiquitination level of Orai1 was detected by immunoprecipitation assay. ns, not significant, * p < 0.05, ** p < 0.01. The data represent three independent experiments with similar results. Unpaired Student's t test was used in a .

Article Snippet: The membranes were then stained with HRP-conjugated secondary antibody (S0001; Affinity Biosciences) at room temperature for another 1 h. Antibodies involved in this subsection are listed below: E-cadherin (14472, mouse anti-human monoclonal antibody, 1:1,000 dilution; Cell Signaling Technology), N-cadherin (22018-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), α-SMA (14395-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), vimentin (10366-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), Orai1 (66223-1-Ig, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), Stim1 (5668, rabbit anti-human monoclonal antibody, 1:1,000 dilution; Cell Signaling Technology), SGK1 (23394-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), PDK1 (17086-1-AP, rabbit anti-human polyclonal antibody, 1:1,000 dilution; Proteintech), GAPDH (60004-1-Ig, mouse anti-human monoclonal antibody, 1:1,000 dilution; Proteintech), and Ub (sc-8017, mouse anti-human monoclonal antibody, 1:1,000 dilution; Santa Cruz Biotechnology).

Techniques: Ubiquitin Proteomics, Incubation, Quantitative RT-PCR, Expressing, Western Blot, Immunoprecipitation

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