Journal: Advanced Science

Article Title: Calcium–Collagen Coupling is Vital for Biomineralization Schedule

doi: 10.1002/advs.202100363

Figure Lengend Snippet: Alteration of the Col1 biosynthesis caused consistent changes in ER Ca 2+ concentrations. A) Schematic illustration. Based on quantitative consistency of Ca 2+ and collagen, BMSCs underwent osteogenic induction by OM, and then the collagen expression was inhibited and recovered, followed by determination of Ca 2+ and collagen changes. Lv‐Col1 indicates lentiviral Col1 plasmids. B,C) The inhibitory effects of FT011 and the retrieval effects of Lv‐Col1 in dose‐dependent manner. N = 5. D) Fura‐2‐AM fluorescence changes. Scale bar = 10 µm. Histogram shows the fluorescence intensity (arbitrary units). N = 9. E) Cytosolic Ca 2+ changes tested by Fluo‐4‐AM (Fluo 4) (presented in Δ F/F ). Error bars represent ± SEM (control group, grey line, N = 30; inhibitory group, red line, N = 35; retrieval group, blue line, N = 30). Bar chart showing the area under the curves (AUC). N = 5. F) The D1ER fluorescence images. Scale bar = 10 µm. The relative basal FRET ratio (FRET/CFP) of D1ER. N = 6. G) STIM1 localization. Arrowheads = STIM1 puncta. Scale bar = 5 µm. F P / F TOT is calculated as the ratio of fluorescence intensity in the peripheral region ( F P ) to the total cell fluorescence ( F TOT ). N = 5. H) STEM‐EDX elemental mapping and unstained immune‐TEM images. Insets are high magnification of selected areas. Scale bar = 500 nm and = 200 nm in insets. Unstained sections are pseudo‐colored. Arrows = Col1. I,J) The comparison of Col1 positive dots per cell and elemental compositions (at%) among regions containing ER. N = 6. K,L) The comparison of sectional size of dilated ER and the percentage of cells bearing dilated ER. N = 10. Cells in this figure are osteogenic induced for 3 days and then treated for 2h, with or without transfection. All of the experiments are performed at least three times. * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.0001, ns , no significant difference.

Article Snippet: [ ] To inhibit the expression of type I collagen, BMSCs were transfected with empty lentiviral vector and treated with the collagen inhibitor, FT011 (HY‐100495, MedChemExpress, USA) for 2 h. [ ] To stimulate STIM1 redistribution, 10 × 10 −6 m TG was used for 10 min. To chelate cytosolic Ca 2+ , 40 × 10 −6 m 1,2‐bis(o‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA, HY‐100168, MedChemExpress, USA) or an equivalent amount of DMSO was added to the OM solution.

Techniques: Expressing, Fluorescence, Control, Comparison, Transfection

Journal: Advanced Science

Article Title: Calcium–Collagen Coupling is Vital for Biomineralization Schedule

doi: 10.1002/advs.202100363

Figure Lengend Snippet: Knockdown of TRAM2 disrupted the connection between Ca 2+ and Col1. A) Schematic illustration. After bioinformatics analysis and preliminary screening, TRAM2‐knockdown BMSCs underwent osteogenic induction by OM, finding the inhibition and depletion of Ca 2+ and collagen, with the coupling disrupted. B) The D1ER fluorescence images. Scale bar = 10 µm. The relative basal FRET ratio (FRET/CFP) of D1ER. N = 6. C) STIM1 localization. Arrowheads = STIM1 puncta. Scale bar = 10 µm. F P / F TOT calculation is performed as mentioned above. N = 5; one‐way ANOVA with Dunnett's multiple comparisons test. D‐E) Col1 and GAPDH (loading control) or CANX (loading control for ER protein) immunoblots of total and ER proteins. N = 4; two‐tailed Student's t ‐test. F) Col1 and GAPDH (loading control) immunoblots of cell (without extracellular matrix) and cytoplasm proteins. N = 4. G) Fura‐2‐AM fluorescence changes. Scale bar = 10 µm. Histogram shows the fluorescence intensity (arbitrary units). N = 9. H) Cytosolic Ca 2+ changes tested by Fluo4 (Δ F/F ) treated in the absence of Ca 2+ . Error bars represent ± SEM (control group, grey line, N = 40; knockdown group, red line, N = 35; + FT011 group, blue line, N = 40; retrieval group, green line, N = 45). Bar chart showing the area under the curves (AUC). N = 5. I) The D1ER fluorescence images. Scale bar = 10 µm. The relative basal FRET ratio (FRET/CFP) of D1ER. N = 6. J) ER Ca 2+ levels were monitored by D1ER in control group, N = 40; knockdown group, N = 35; + FT011 group, N = 40; retrieval group, N = 45, after the stimulation of 100 × 10 −6 m ATP. Error bars represent ± SEM. Histogram shows the average ER Ca 2+ levels in resting cells in each group. N = 10. Lv‐sh indicates TRAM2 knockdown cells. All of the experiments are performed at least three times. * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.0001, ns , no significant difference.

Article Snippet: [ ] To inhibit the expression of type I collagen, BMSCs were transfected with empty lentiviral vector and treated with the collagen inhibitor, FT011 (HY‐100495, MedChemExpress, USA) for 2 h. [ ] To stimulate STIM1 redistribution, 10 × 10 −6 m TG was used for 10 min. To chelate cytosolic Ca 2+ , 40 × 10 −6 m 1,2‐bis(o‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA, HY‐100168, MedChemExpress, USA) or an equivalent amount of DMSO was added to the OM solution.

Techniques: Knockdown, Inhibition, Fluorescence, Control, Western Blot, Two Tailed Test

Journal: Journal of Neuroscience

Article Title: Store-Operated Ca2+ Entry in Sensory Neurons: Functional Role and the Effect of Painful Nerve Injury

doi: 10.1523/jneurosci.5053-10.2011

Figure Lengend Snippet: Figure 6. Immunohistochemical identification of STIM1 distribution. A, Staining for STIM1 (left panels) was uniformly distributed within cytoplasmic areas of cells identified as neuronal profiles through costaining with NeuN (middle panels). Only minimal nonspecific staining was present in fiber tracts. The scale bar applies to each panel. B, Morphometric analysis showed comparableintensity(arbitraryunits,recordedusingstandardizedimageacquisition)ofstain- inginneuronsofallsizes(ANOVA,p0.40).ShownaremeanSEM.C,STIM1(leftpanel)was also found in cellular components identified as satellite glial cells by their expression of glu- tamine synthase (GS) (middle panel). Identically placed arrows in the left and middle panels indicate cellular areas clearly identifiable as satellite glial cell cytoplasm that expresses STIM1. The scale bar applies to each panel.

Article Snippet: After blocking with 5% milk in TBST (Trisbuffered saline plus 0.1% Tween 20), blots were sequentially probed with anti- -Tubulin I mouse monoclonal antibody (1:20,000; Sigma-Aldrich; catalog #T7816), anti-STIM1 rabbit polyclonal antibody (1:500; ProSci; catalog #4119), and anti-Orai1 rabbit polyclonal antibody (1:1000; ProSci, catalog #4281).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Journal of Neuroscience

Article Title: Store-Operated Ca2+ Entry in Sensory Neurons: Functional Role and the Effect of Painful Nerve Injury

doi: 10.1523/jneurosci.5053-10.2011

Figure Lengend Snippet: Figure 5. Identification of molecular components of store-operated Ca 2 channels. A, Western blotting detected Orai1 and STIM1 proteins in lysates from control (C) dorsal root ganglia (DRG), from fourth lumbar DRGs after fifth lumbar spinal nerve ligation (SNL L4), and fromaxotomizedfifthlumbarDRGsafterSNL(SNLL5).AdominantSTIM1bandattheexpected molecularweightisaccompaniedbyacloselycoherentsecondarybandthatothershaveshown to be sensitive to STIM1 knockdown by RNAi (Ong et al., 2007). (Typical of n 3.) B, Quantifi- cation of Western blots as fold difference compared with control showed no effect of injury on Orai1andSTIM1proteinlevels.ShownaremeanSEM.C,Quantificationoftranscriptlevelsby quantitativertPCRalsoshowednoeffectsofinjuryonOrai1andSTIM1expression.ForBandC, the numbers in bars indicate n.

Article Snippet: After blocking with 5% milk in TBST (Trisbuffered saline plus 0.1% Tween 20), blots were sequentially probed with anti- -Tubulin I mouse monoclonal antibody (1:20,000; Sigma-Aldrich; catalog #T7816), anti-STIM1 rabbit polyclonal antibody (1:500; ProSci; catalog #4119), and anti-Orai1 rabbit polyclonal antibody (1:1000; ProSci, catalog #4281).

Techniques: Western Blot, Control, Ligation, Knockdown

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: The Journal of Physiology

Article Title: Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

doi: 10.1113/JP273302

Figure Lengend Snippet: A , western blots and mean data confirm that two different STIM1 shRNA sequences (shRNA1 and shRNA2) reduced STIM1 expression (three primary rabbit portal vein VSMC culture preparations; ** P < 0.01). B , traces and mean I / V relationships showing that peak amplitude of store‐operated whole‐cell TRPC1‐based currents were greatly reduced at all membrane potentials tested following transduction of rabbit portal vein VSMCs with shRNA sequences compared to scrambled shRNA sequences. In the presence of scrambled sequences, store‐operated TRPC1‐based currents were inhibited by an anti‐STIM1 antibody ( n = 6). C , recordings and mean data showing that BAPTA‐AM‐evoked TRPC1‐based SOCs were reduced by shRNA sequences targeting STIM1 compared to scrambled shRNA in VSMCs ( n = 6; *** P < 0.001).

Article Snippet: Rabbit anti‐STIM1 antibody against the N‐terminal EF hand (11565‐1‐AP) was obtained from Proteintech (Chicago, IL, USA), mouse anti‐GOK/STIM1 (610954) against the N‐terminal EF hand was obtained from BD Biosciences (Oxford, UK) and mouse anti‐STIM1 antibody against the C‐terminal region (SC‐66173) was obtained from Santa Cruz.

Techniques: Western Blot, shRNA, Expressing, Transduction

Journal: The Journal of Physiology

Article Title: Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

doi: 10.1113/JP273302

Figure Lengend Snippet: A , co‐immunoprecipitation of rabbit portal vein tissue lysates with anti‐phosphorylated serine (pSer) and threonine (pThr) antibodies followed by western blotting (WB) with an anti‐TRPC1 antibody shows that basal TRPC1 phosphorylation is increased by pre‐treatment with BAPTA‐AM for 10 min, and that this increase was reduced by STIM1 shRNA1 and coapplication of GF109203X. B , mean relative band densities normalized to BAPTA‐AM bands (three different tissue lysate preparations; * P < 0.05, ** P < 0.01). [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Rabbit anti‐STIM1 antibody against the N‐terminal EF hand (11565‐1‐AP) was obtained from Proteintech (Chicago, IL, USA), mouse anti‐GOK/STIM1 (610954) against the N‐terminal EF hand was obtained from BD Biosciences (Oxford, UK) and mouse anti‐STIM1 antibody against the C‐terminal region (SC‐66173) was obtained from Santa Cruz.

Techniques: Immunoprecipitation, Western Blot

Journal: The Journal of Physiology

Article Title: Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

doi: 10.1113/JP273302

Figure Lengend Snippet: A , image from a single rabbit portal vein VSMC showing that in control conditions the location of GFP‐PLCδ1‐PH‐mediated signals (measured as relative fluorescence units; RFU) was predominantly expressed at the PM (black). In the same cell, pre‐treatment with BAPTA‐AM for 10 min induced translocation of signals to the cytosol (blue), and coapplication of U73122 for 5 min reversed these cytosolic signals back to the PM (orange). Graphs of RFU of line scans for the region denoted along the white dotted lines show GFP‐PLCδ1‐PH signals across the cell width. Mean F m / F c ratios of GFP‐PLCδ1‐PH‐mediated signals represent 20 cells from three different experiments ( *** P <0.001). B and C , images and mean data showing that transduction of rabbit portal vein VSMCs with either STIM1 shRNA1 or shRNA2 sequences prevented BAPTA‐AM inducing translocation of GFP‐PLCδ1‐PH signals to the cytosol. Under both these conditions, application of noradrenaline for 5 min (red, applied in the presence of 1 μ m Wortmannin to prevent cell contraction) was still able to induce translocation of GFP‐PLCδ1‐PH signals from the PM to the cytosol (20 cells for each STIM1 shRNA sequence from three different experiments; *** P < 0.01).

Article Snippet: Rabbit anti‐STIM1 antibody against the N‐terminal EF hand (11565‐1‐AP) was obtained from Proteintech (Chicago, IL, USA), mouse anti‐GOK/STIM1 (610954) against the N‐terminal EF hand was obtained from BD Biosciences (Oxford, UK) and mouse anti‐STIM1 antibody against the C‐terminal region (SC‐66173) was obtained from Santa Cruz.

Techniques: Fluorescence, Translocation Assay, Transduction, shRNA, Sequencing

Journal: The Journal of Physiology

Article Title: Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

doi: 10.1113/JP273302

Figure Lengend Snippet: A , bath application of noradrenaline evoked TRPC1 channel activity in a concentration‐dependent manner in cell‐attached patches from WT mesenteric artery VSMCs held at −80 mV, which were reduced in VSMCs expressing STIM1 shRNA1 and shRNA2 sequences compared to scrambled shRNA. B , mean data showing the inhibitory actions of STIM1 shRNA1 and shRNA2 on noradrenaline‐evoked TRPC1 channel activity (at least six patches in which every concentration of noradrenaline tested was cumulatively applied; ** P < 0.01, *** P < 0.001). C , PLA images from single WT VSMCs showing that application of noradrenaline for 5 min induced fluorescence signals (red), which indicated interactions between TRPC1 and STIM1, STIM1 and Gαq, and STIM1 and PLCβ1 predominantly at the PM.

Article Snippet: Rabbit anti‐STIM1 antibody against the N‐terminal EF hand (11565‐1‐AP) was obtained from Proteintech (Chicago, IL, USA), mouse anti‐GOK/STIM1 (610954) against the N‐terminal EF hand was obtained from BD Biosciences (Oxford, UK) and mouse anti‐STIM1 antibody against the C‐terminal region (SC‐66173) was obtained from Santa Cruz.

Techniques: Activity Assay, Concentration Assay, Expressing, shRNA, Fluorescence

Journal: The Journal of Physiology

Article Title: Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

doi: 10.1113/JP273302

Figure Lengend Snippet: A and B , images from two different rabbit portal vein VSMCs showing TRPC1 (green) and STIM1 (red) staining. Changes in the relative fluorescence of TRPC1 and STIM1 staining across the cell width were determined from the line scan (dotted white line). In a resting cell ( A ), TRPC1 staining was predominantly present at the PM, whereas labelling for STIM1 was located within the cytosol. In a cell treated with BAPTA‐AM for 10 min ( B ), both TRPC1 and STIM1 were located at the PM in discrete puncta. Inset: co‐localization between TRPC1 and STIM1 staining (yellow) at the PM. C and D , PLA images showing BAPTA‐AM induced fluorescence signals between STIM1 and TRPC1 which were predominantly at the PM in rabbit portal vein and mice mesenteric artery VSMCs. E , immunocytochemical image showing that, in TRPC1 −/− mesenteric artery VSMCs, BAPTA‐AM induced translocation of STIM1 from the cytosol to the PM where it produced uniform, non‐puncta‐like staining.

Article Snippet: Rabbit anti‐STIM1 antibody against the N‐terminal EF hand (11565‐1‐AP) was obtained from Proteintech (Chicago, IL, USA), mouse anti‐GOK/STIM1 (610954) against the N‐terminal EF hand was obtained from BD Biosciences (Oxford, UK) and mouse anti‐STIM1 antibody against the C‐terminal region (SC‐66173) was obtained from Santa Cruz.

Techniques: Staining, Fluorescence, Translocation Assay, Produced

Journal: The Journal of Physiology

Article Title: Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

doi: 10.1113/JP273302

Figure Lengend Snippet: A , western blots showing that pre‐treatment with BAPTA‐AM for 10 min induced associations between TRPC1 and STIM1, Gαq and PLCβ1, which were reduced by STIM1 shRNA1. Primary cultured rabbit portal vein VSMC lysates were initially immunoprecipitated (IP) with anti‐TRPC1 antibodies and were then western blotted (WB) with anti‐STIM1, anti‐Gαq or anti‐PLCβ antibodies. B , mean data for relative band intensities of BAPTA‐AM‐evoked interactions with TRPC1 shown in A (three different cell lysates; * P < 0.05). C , application of BAPTA‐AM for 10 min did not alter interactions between TRPC6 and STIM1 (left) or change expression levels of TRPC6 (right) in rabbit portal vein tissue lysates. D , primary cultured WT mesenteric artery VSMCs: BAPTA‐AM evoked interactions between STIM1 and TRPC1, Gαq and PLCβ1, which were absent in cell lysates from TRPC1 −/− VSMCs.

Article Snippet: Rabbit anti‐STIM1 antibody against the N‐terminal EF hand (11565‐1‐AP) was obtained from Proteintech (Chicago, IL, USA), mouse anti‐GOK/STIM1 (610954) against the N‐terminal EF hand was obtained from BD Biosciences (Oxford, UK) and mouse anti‐STIM1 antibody against the C‐terminal region (SC‐66173) was obtained from Santa Cruz.

Techniques: Western Blot, Cell Culture, Immunoprecipitation, Expressing

Journal: The Journal of Physiology

Article Title: Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

doi: 10.1113/JP273302

Figure Lengend Snippet: A , PLA images from WT mesenteric artery VSMCs showing that BAPTA‐AM induced interactions between STIM and Gαq, and also between STIM1 and PLCβ1. B , BAPTA‐AM‐evoked interactions between STIM and Gαq, and STIM1 and PLCβ1 were absent in TRPC1 −/− VSMCs.

Article Snippet: Rabbit anti‐STIM1 antibody against the N‐terminal EF hand (11565‐1‐AP) was obtained from Proteintech (Chicago, IL, USA), mouse anti‐GOK/STIM1 (610954) against the N‐terminal EF hand was obtained from BD Biosciences (Oxford, UK) and mouse anti‐STIM1 antibody against the C‐terminal region (SC‐66173) was obtained from Santa Cruz.

Techniques:

Journal: The Journal of Physiology

Article Title: Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

doi: 10.1113/JP273302

Figure Lengend Snippet: A – C , application of BAPTA‐AM for 10 min induced interactions between TRPC1 and PLCβ1 in rabbit portal vein VSMCs measured using PLA that were reduced by expression of PLCβ1 shRNA1 and shRNA2 sequences, whereas associations between TRPC1 and STIM1 were unaffected (three different preparations; * P < 0.05). D and E , BAPTA‐AM‐induced interactions between TRPC1 and PLCβ1 measured using co‐immunoprecipitation were reduced by expression of PLCβ1 shRNA1 and shRNA2 sequences, whereas associations between TRPC1 and STIM1 were unaffected (three different rabbit portal vein cell lysates; * P < 0.05).

Article Snippet: Rabbit anti‐STIM1 antibody against the N‐terminal EF hand (11565‐1‐AP) was obtained from Proteintech (Chicago, IL, USA), mouse anti‐GOK/STIM1 (610954) against the N‐terminal EF hand was obtained from BD Biosciences (Oxford, UK) and mouse anti‐STIM1 antibody against the C‐terminal region (SC‐66173) was obtained from Santa Cruz.

Techniques: Expressing, Immunoprecipitation

Journal: The Journal of Physiology

Article Title: Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

doi: 10.1113/JP273302

Figure Lengend Snippet: A , original trace showing that a store‐operated whole‐cell current from a WT mesenteric artery VSMC was inhibited by bath application of a N‐terminal but not a C‐terminal STIM1 antibody. B , representative recording showing that BAPTA‐AM‐evoked single channel activity in an inside‐out patch from a WT VSMC held at −80 mV was inhibited by bath application of a C‐terminal but not a N‐terminal STIM1 antibody. C , representative images from two different VSMCs treated with Triton, in which both N‐terminal and C‐terminal STIM1 antibodies WT identified translocation of STIM1 signalling (red) from the cytosol to the PM following treatment with BAPTA‐AM for 10 min. D , representative images from two different WT VSMCs not treated with Triton, in which N‐terminal, nor C‐terminal STIM1 antibodies identified STIM1 staining in unstimulated cells, and only the N‐terminal antibody revealed STIM1 staining at the PM following treatment with BAPTA‐AM.

Article Snippet: Rabbit anti‐STIM1 antibody against the N‐terminal EF hand (11565‐1‐AP) was obtained from Proteintech (Chicago, IL, USA), mouse anti‐GOK/STIM1 (610954) against the N‐terminal EF hand was obtained from BD Biosciences (Oxford, UK) and mouse anti‐STIM1 antibody against the C‐terminal region (SC‐66173) was obtained from Santa Cruz.

Techniques: Activity Assay, Translocation Assay, Staining

Journal: The Journal of Physiology

Article Title: Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

doi: 10.1113/JP273302

Figure Lengend Snippet: A , in resting VSMCs SR stores are filled with Ca 2+ , TRPC1‐based SOCs do not interact with Gαq, PLCβ1 or STIM1, and the channels are in a closed state. B , following store depletion of the SR, STIM1 proteins (red) are activated and translocated from the SR into the PM, where they interact with TRPC1. Following translocation, the N‐terminal EF hand of STIM1, which acts as a Ca 2+ sensor within the SR, is exposed on the external surface of the cell, whereas the C‐terminal region is maintained within the cytosol. C , formation of store‐operated STIM1‐TRPC1 interactions enable the binding of Gαq and PLCβ1, which stimulate PLC activity, leading to PIP 2 hydrolysis, formation of DAG, PKC stimulation, phosphorylation of TRPC1 subunits and channel opening.

Article Snippet: Rabbit anti‐STIM1 antibody against the N‐terminal EF hand (11565‐1‐AP) was obtained from Proteintech (Chicago, IL, USA), mouse anti‐GOK/STIM1 (610954) against the N‐terminal EF hand was obtained from BD Biosciences (Oxford, UK) and mouse anti‐STIM1 antibody against the C‐terminal region (SC‐66173) was obtained from Santa Cruz.

Techniques: Translocation Assay, Binding Assay, Activity Assay