rabbit anti ano1  (Proteintech)

Journal: The Journal of Cell Biology

Article Title: Ca 2+ tunneling architecture and function are important for secretion

doi: 10.1083/jcb.202402107

Figure Lengend Snippet: CaTAr1 effects on tunneling and localization of ANO1–GFP and mCh-STIM1. (A) Dose-dependent inhibition of tunneling by CaTAr1. The amplitude of the tunneling signal is plotted as a function of the GFP signal normalized from minimum to maximum signal in each dish. The data is sorted in bins of 10%. (B) The relative localization of ANO1–GFP and mCh-STIM1 in NCL cells after store depletion is similar to the endogenous ANO1 channel and suggests the separation of ANO1 from the STIM1 cluster. (C) TIRF imaging of ANO1–GFP and mCh-STIM1 during store depletion with CPA confirmed the separation of the two proteins at the PM. Ctr and CPA images are from the same region of interest.

Article Snippet: The following primary antibodies were used at a 1:500 dilution: SERCA2 (NB300-581; Novus Biologicals), STIM1 (5668S; Cell Signaling and MAI-19451; Thermo Fisher Scientific), KRAP (14157-1-AP; Thermo Fisher Scientific), NFAT1 (5861S; Cell Signaling), and ANO1 (14476; Cell Signaling).

Techniques: Inhibition, Imaging

Journal: The Journal of Cell Biology

Article Title: Ca 2+ tunneling architecture and function are important for secretion

doi: 10.1083/jcb.202402107

Figure Lengend Snippet: Tunneling in NCL-SG3 cells. (A) Localization of ANO1 by immunocytochemistry and mCh-STIM1 before (Ctr) and after store depletion (Tg). An intensity plot performed along a line (arrow) crossing a STIM1 cluster illustrates the separation of STIM1 and ANO1 at the PM focal plane. (B–D) Comparative localization at the PM plane of mCh-STIM1 with ANO1–GFP and the endogenous ANO1 protein (ANO1–Ig) following store depletion. As indicated by the PCC and the peak-to-peak distances the two proteins do not colocalize laterally but are localized at the same optical plane (z distance) ( n = 8–18; one-way ANOVA, P < 0.0001 for B and C, P = 0.7 for D). (E) Intracellular Ca 2+ elevation induced by trypsin application in NCL-SG3 cells loaded with Fluo4-AM. The application of the SOCE inhibitor BTP-2 (10 μM) reduces the amplitude and the duration of Ca 2+ release ( n = 379/402; unpaired t test, P < 0.0001). (F) Confocal images of NCL-SG3 cells expressing the Cl − sensor mbYFPQS and mCh-STIM1 after store depletion. The orthogonal section through the cell indicates the PM localization of the chloride sensor. (G) Kymographs were measured during a tunneling event on cells expressing mbYFPQS and mCh-STIM1. The line passes through a SOCE cluster and an adjacent cell appendage labeled by mbYFPQS. The changes in Cl − concentration (upper) are distal from the STIM1 cluster, which indicates the Ca 2+ entry point. (H) Time course of the amplitude of the Cl − signal induced by Ca 2+ tunneling from NCL-SG3 cells in the same dish that either do not show any CaTAr2 expression (Ctr) and cells expressing CaTAr2. (I) Violin plots summarizing the amplitude of the Cl − signal 3 min after trypsin and Ca 2+ addition to stimulate tunneling ( n = 32–33; unpaired t test, P = 0.0004).

Article Snippet: The following primary antibodies were used at a 1:500 dilution: SERCA2 (NB300-581; Novus Biologicals), STIM1 (5668S; Cell Signaling and MAI-19451; Thermo Fisher Scientific), KRAP (14157-1-AP; Thermo Fisher Scientific), NFAT1 (5861S; Cell Signaling), and ANO1 (14476; Cell Signaling).

Techniques: Immunocytochemistry, Expressing, Labeling, Concentration Assay

Journal: The Journal of Cell Biology

Article Title: Ca 2+ tunneling architecture and function are important for secretion

doi: 10.1083/jcb.202402107

Figure Lengend Snippet: CaTAr2 and ANO1–GFP in NCL cells. (A) Airy scan images of the Co-expression of CaTAr2 and ANO1–GFP in NCL cells indicate the partial overlap between the two signals. (B) The colocalization is not influenced by store depletion. (C) Relative intensities along a virtual line scan (white arrows in A) reveal the colocalization of ANO1 and CaTAr2 but also enrichment in ANO1 at the edge of the CaTAr2-rich ER patch.

Article Snippet: The following primary antibodies were used at a 1:500 dilution: SERCA2 (NB300-581; Novus Biologicals), STIM1 (5668S; Cell Signaling and MAI-19451; Thermo Fisher Scientific), KRAP (14157-1-AP; Thermo Fisher Scientific), NFAT1 (5861S; Cell Signaling), and ANO1 (14476; Cell Signaling).

Techniques: Expressing

Journal: eLife

Article Title: Involvement of TRPV4 in temperature-dependent perspiration in mice

doi: 10.7554/eLife.92993

Figure Lengend Snippet: ( A ) TRPV4 signals in the secretory coil in the deep dermis with a relatively straight duct opening to the skin surface. ( B ) Localization of TRPV4 (green), cytokeratin 8 (CK8; yellow), and anoctamin 1 (ANO1; magenta) in the skin. ( C–E ) Highly magnified Airyscan super-resolution images of the sweat duct ( C ) and secretory portion ( D, E ). ( C ) TRPV4 localizes to the basal cells of the bilayered sweat duct. Ductal lumen: L. ( D ) Secretory gland showing labeling for TRPV4 and calcitonin gene-related peptide (CGRP). ( E ) Secretory gland with conspicuous TRPV4 labeling in myoepithelial cells (M) and secretory cells. TRPV4 clearly colocalizes with aquaporin-5 (AQP5), F-actin, and ANO1 at the luminal side of the secretory cells. Arrows indicate the glandular lumen. DIC, differential interference contrast; nuclei: DAPI. Scale bar: 50 μm ( A, B ). 5 μm ( C–E ).

Article Snippet: Sections were then incubated overnight at 4°C with the primary antibodies: guinea pig anti-TRPV4 (2 μg/mL) , rabbit anti-ANO1 (1:100, Abcam, ab53213), rabbit anti-AQP5 (1:200, Millipore, 178615), rabbit anti-CGRP (1:2000, Amersham International, RPN.1842), rat anti-cytokeratin 8 (CK8) (1:100, Millipore, MABT329).

Techniques: Labeling

Journal: eLife

Article Title: Involvement of TRPV4 in temperature-dependent perspiration in mice

doi: 10.7554/eLife.92993

Figure Lengend Snippet:

Article Snippet: Sections were then incubated overnight at 4°C with the primary antibodies: guinea pig anti-TRPV4 (2 μg/mL) , rabbit anti-ANO1 (1:100, Abcam, ab53213), rabbit anti-AQP5 (1:200, Millipore, 178615), rabbit anti-CGRP (1:2000, Amersham International, RPN.1842), rat anti-cytokeratin 8 (CK8) (1:100, Millipore, MABT329).

Techniques: Recombinant, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

doi: 10.1101/2023.06.07.544093

Figure Lengend Snippet: A, Representative images of western blot analyses of the effect of the pharmacological inhibitor of ANO1, T16A inh -A01 (T16Ai) on myofibroblast differentiation. Serum-starved (48 hr) HLF were pretreated with 30 μM T16Ai or vehicle control for 1hr, followed by treatment with TGF-β (1 ng/ml) for 48 hr. Cell lysates were then probed for immunoreactivity of anactomin-1 (ANO1), collagen 1A1 (Col1A1), fibronectin (FN), and smooth muscle α-actin (SMA). B, Quantification of ANO1, Col1A1, FN, and SMA immunoreactivities. Data are mean values ± SD. *p<0.05, **p<0.01, ***p<0.001, vs. TGFβ group, Kruskal-Wallis test with Dunn’s post hoc correction. Brace brackets, ### p<0.001; #### , p<0.0001, are the results of preplanned comparisons of TGF-β effect with and without ANO1 inhibitor, one-population t-test.

Article Snippet: Antibodies against ANO1 (14476, 1,000×), Smad2 (L1603, 1,000×), phospho-Smad2-Ser465/467 (138D4, 1,000×), phospho-MLC-Ser19 (3671, 1,000×) and phospho-AKT-Ser473 (9271, 1,000×) were from Cell Signaling Technology.

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

doi: 10.1101/2023.06.07.544093

Figure Lengend Snippet: A, Representative images of western blot analyses of the effect of siRNA targeting ANO1 (siANO1) on myofibroblast differentiation. HLF were transfected with siRNA targeting ANO1 (siANO1) or control RNA (siCont) for 24 hrs, serum starved for 48 hr, and treated with TGF-β or vehicle control for 48 hrs. Cell lysates were then probed for immunoreactivity of anactomin-1 (ANO1), collagen 1A1 (Col1A1), fibronectin (FN), and smooth muscle α-actin (SMA). B, Quantification of ANO1, Col1A1, FN, and SMA immunoreactivity. Data are mean values ±SD. *p<0.05, **p<0.01, ***p<0.001, vs. TGFβ group, Kruskal-Wallis test with Dunn’s post hoc correction. Brace brackets, ## p<0.01 ### p<0.001; #### , p<0.0001, are the results of preplanned comparisons of TGF-β effect in cells treated with either control siRNA (siC) or siANO1, one-population t-test.

Article Snippet: Antibodies against ANO1 (14476, 1,000×), Smad2 (L1603, 1,000×), phospho-Smad2-Ser465/467 (138D4, 1,000×), phospho-MLC-Ser19 (3671, 1,000×) and phospho-AKT-Ser473 (9271, 1,000×) were from Cell Signaling Technology.

Techniques: Western Blot, Transfection, Control

Journal: bioRxiv

Article Title: Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

doi: 10.1101/2023.06.07.544093

Figure Lengend Snippet: A, Representative images of western blot analyses of the pharmacological effect of the ANO1 inhibitor, T16Ainh-A01 (T16Ai), on intracellular signaling pathways in HLF. Serum-starved (48 hr) cells were pretreated for 1 hr with T16Ai, followed by treatment with TGF-β (1 ng/ml) for either 30 min (p-Smad2, Smad2), or 48 hr (p-MLC, MLC, p-AKT, AKT). B, Quantification of p-MLC and p-AKT immunoreactivities in T16Ai-treated cells. Data represent the mean immunoreactivity values ±SD normalized to TGF-β group within the same experiment. *p<0.05, **p<0.01, vs. TGF-β group, Kruskal-Wallis test with Dunn’s post hoc correction. Brace brackets, #p<0.05; ##, p<0.01, are the results of preplanned comparisons of the TGFβ effect in cells treated with or without T16Ai, one population t-test. C, Representative images of western blot analyses of the effects of the siRNA targeting ANO1 (siANO1) on intracellular signaling pathways in HLF. HLF cells were transfected with the siRNA targeting ANO1 (siANO1) or control RNA (siCont) for 24 hrs, serum starved for 48 hrs, and treated with TGFβ (1 ng/ml) for either 30 min (p-Smad2, Smad2), or for 48 hr (p-MLC, MLC, p-AKT, AKT). D, Quantification of p-MLC and p-AKT immunoreactivities in siCont and siANO1-treated cells. Data represent mean immunoreactivity values ±SD normalized to β-actin loading control and TGF-β group within the same experiment. * *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, vs. TGF-β group, Kruskal-Wallis test with Dunn’s post hoc correction. Brace brackets, ###p<0.001; ####, p<0.0001, are the results of preplanned comparisons of the TGF-β effect in cells treated with control siRNA (siC) or siANO1, one population t-test.

Article Snippet: Antibodies against ANO1 (14476, 1,000×), Smad2 (L1603, 1,000×), phospho-Smad2-Ser465/467 (138D4, 1,000×), phospho-MLC-Ser19 (3671, 1,000×) and phospho-AKT-Ser473 (9271, 1,000×) were from Cell Signaling Technology.

Techniques: Western Blot, Protein-Protein interactions, Transfection, Control

Journal: bioRxiv

Article Title: Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

doi: 10.1101/2023.06.07.544093

Figure Lengend Snippet: A, Representative images of western blot analyses of the effects of siRNA targeting WNK1 (siWNK1) and the WNK kinase inhibitor WNK463 on myofibroblast markers and intracellular signaling pathways in HLF. HLF were transfected with siRNA targeting ANO1 (siANO1) or control RNA (siCont) for 24 hrs, serum starved for 48 hrs, pretreated with or without WNK1 inhibitor WNK463 (10 μM) for 1 hr, followed by treatment with TGF-β (1 ng/ml) or vehicle control for 48 hrs. Cell lysates were then probed for the immunoreactivity of WNK1, collagen 1A1 (Col1A1), fibronectin (FN), smooth muscle α-actin (SMA), p-MLC, MLC, p-AKT, and AKT. B, Quantification of the immunoreactivity of WNK, Col1A1, FN, SMA, p-MLC, and p-AKT. Data are mean IR values ±SD normalized to β-actin loading control and TGFβ group within the same experiment. *p<0.05, **p<0.01, ***p<0.001, vs. TGF-β group, Kruskal-Wallis test with Dunn’s post hoc correction. Brace brackets, #p<0.05; ##, p<0.01, are the results of preplanned comparisons of the TGFβ effect in cells treated with siWNK1 or the WNK1 inhibitor WNK463, t-test.

Article Snippet: Antibodies against ANO1 (14476, 1,000×), Smad2 (L1603, 1,000×), phospho-Smad2-Ser465/467 (138D4, 1,000×), phospho-MLC-Ser19 (3671, 1,000×) and phospho-AKT-Ser473 (9271, 1,000×) were from Cell Signaling Technology.

Techniques: Western Blot, Protein-Protein interactions, Transfection, Control

Journal: bioRxiv

Article Title: Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

doi: 10.1101/2023.06.07.544093

Figure Lengend Snippet: A, Representative images of western blot analyses of the effects of siRNA targeting WNK1 (siWNK1) or ANO1 (siANO1) on myofibroblast differentiation and intracellular signaling in IPF-HLF. Cells were transfected with siRNA targeting WNK1 (siWNK1), ANO1 (siANO1) or control RNA (siCont) for 24 hrs, serum starved for 48 hrs, followed by treatment with TGF-β (1 ng/ml) or vehicle control for 48 hrs. Cell lysates were then probed for the immunoreactivity of WNK1, collagen 1A1 (Col1A1), fibronectin (FN), smooth muscle α-actin (SMA), p-MLC, MLC, p-AKT, and AKT. B, Quantification of the immunoreactivity of WNK, Col1A1, FN, SMA, p-MLC, and p-AKT. Data are mean values ±SD normalized to TGF-β group within the same experiment. *p<0.05, **p<0.01, vs. TGF-β group, Kruskal-Wallis test with Dunn’s post hoc correction. Brace brackets, #p<0.05; ##, p<0.01, are the results of preplanned comparisons of the TGF-β effect in cells treated with or without T16Ai, one population t-test.

Article Snippet: Antibodies against ANO1 (14476, 1,000×), Smad2 (L1603, 1,000×), phospho-Smad2-Ser465/467 (138D4, 1,000×), phospho-MLC-Ser19 (3671, 1,000×) and phospho-AKT-Ser473 (9271, 1,000×) were from Cell Signaling Technology.

Techniques: Western Blot, Transfection, Control

Journal: bioRxiv

Article Title: Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

doi: 10.1101/2023.06.07.544093

Figure Lengend Snippet: Human lung fibroblasts were serum starved in 0.1% FBS for 48 hr, followed by treatment with TGF-β (1 ng/ml) for 24 or 48 hrs, as indicated. A, ANO1 mRNA expression after 24-hr treatment with TGF-β as determined by RT-qPCR. Data are mean values ±SD. ***p<0.001, t-test. B, Western blot analysis and quantitation of ANO1 protein expression in response TGF-β. Data are mean values normalized to control cells ±SD. *, p<0.05, ***p<0.001, Kruskal-Wallis test with Dunn’s correction for multiple comparisons. C, Effect of the TGFBRII inhibitor, SB-431542 (5 μM, 30 minutes pre-incubation) on TGF-β-induced ANO1 protein expression (48 hrs). Shown are representative images of ANO1 immunoreactivity and quantification. Data are mean immunoreactivity values normalized to TGF-β-treated cells ±SD. ***p<0.001, one-population t-test with Bonferroni correction for multiple comparisons.

Article Snippet: Antibodies against ANO1 (14476, 1,000×), Smad2 (L1603, 1,000×), phospho-Smad2-Ser465/467 (138D4, 1,000×), phospho-MLC-Ser19 (3671, 1,000×) and phospho-AKT-Ser473 (9271, 1,000×) were from Cell Signaling Technology.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Quantitation Assay, Control, Incubation

Journal: bioRxiv

Article Title: Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

doi: 10.1101/2023.06.07.544093

Figure Lengend Snippet: A, Representative immunohistochemical images of non-fibrotic (NF) lungs and fibrotic areas of the IPF lungs. Sequential sections of the same lung were probed with either ANO1 antibody or IgG control. Scale bars = 200 μm. B, High magnification immunofluorescence images of section from an IPF lung, stained for ANO1 (green) and smooth muscle a-actin (SMA, red), and counterstained with DAPI (blue). Scale bars = 20 μm. Shown are representative images of lung sections from two IPF individuals. Note that we did not expect stains precisely overlapping because of membrane localization of ANO1 and cytosolic localization of ANO1.

Article Snippet: Antibodies against ANO1 (14476, 1,000×), Smad2 (L1603, 1,000×), phospho-Smad2-Ser465/467 (138D4, 1,000×), phospho-MLC-Ser19 (3671, 1,000×) and phospho-AKT-Ser473 (9271, 1,000×) were from Cell Signaling Technology.

Techniques: Immunohistochemical staining, Control, Immunofluorescence, Staining, Membrane

Journal: bioRxiv

Article Title: Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

doi: 10.1101/2023.06.07.544093

Figure Lengend Snippet: A, Kinetics of 36 Cl accumulation in HLF. Cl uptake was measured as radiotracer accumulation after adding 36 Cl into cell culture medium. Serum-starved HLF cells were pretreated for 30 min with either vehicle or a combination of 1 mM DIDS plus 20 μM bumetanide. The same inhibitors were present in transport assay media. Data are mean values ±SD in three experiments. ****p<0.0001, repeated measures ANOVA; inhibitors vs. control. B, Effect of the ANO1 inhibitor, T16Ainh-A01 (T16Ai), on intracellular Cl levels in control and TGF-β-treated HLF. Intracellular Cl levels were measured as the steady state 36 Cl accumulation and normalized to the number of cells per well as described in methods. T16Ainh-A01 (30 μM) was applied 1 hr before and was present during 36 Cl transport assay. Data represent mean values ±SD from seven independent experiments in two HLF passages. *p<0.01, **p<0.01; ***p<0.001; One-way ANOVA with Tukey post hoc correction for multiple comparisons. C, The effect of siRNA-mediated ANO1 knockdown on intracellular Cl levels in control and TGFβ-treated primary human lung fibroblasts. HLF cells were transfected with the siRNA targeting ANO1 (siANO1) or the negative control RNA (siCont) for 24 hrs, serum starved for 48 hrs, and treated with TGF-β or vehicle control for 48 hrs. The steady-state 36 Cl accumulation was measured and normalized as in panel B. Data represent mean values ±SD of eight independent experiments performed in two HLF passages. **p<0.01, ***p<0.001, One-way ANOVA with Tukey post hoc correction for multiple comparisons.

Article Snippet: Antibodies against ANO1 (14476, 1,000×), Smad2 (L1603, 1,000×), phospho-Smad2-Ser465/467 (138D4, 1,000×), phospho-MLC-Ser19 (3671, 1,000×) and phospho-AKT-Ser473 (9271, 1,000×) were from Cell Signaling Technology.

Techniques: Cell Culture, Transport Assay, Control, Knockdown, Transfection, Negative Control

Journal: bioRxiv

Article Title: Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

doi: 10.1101/2023.06.07.544093

Figure Lengend Snippet: The left part of the diagram illustrates the canonical model of the TGF-β-induced myofibroblast differentiation involving Smad signaling. The right part of the diagram depicts novel mechanism which this study suggests. (i) TGF-β upregulates the expression of ANO1 and elevates intracellular [Cl ]. (ii) Increased [Cl ]i stabilizes autoinhibitory conformation of WNK1 and prompts its association with and activation of mTORC2. (iii) mTORC2 activates AKT and RhoA pathways. (iv) AKT and RhoA promote myofibroblast differentiation via downstream signaling mechanisms, also including P-MLC. (v) WNK1 signaling forms a feedforward mechanism for ANO expression. Yellow color represents signaling molecules whose contribution was tested in this study. Prepared with BioRender.

Article Snippet: Antibodies against ANO1 (14476, 1,000×), Smad2 (L1603, 1,000×), phospho-Smad2-Ser465/467 (138D4, 1,000×), phospho-MLC-Ser19 (3671, 1,000×) and phospho-AKT-Ser473 (9271, 1,000×) were from Cell Signaling Technology.

Techniques: Expressing, Activation Assay

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 1. Pharmacological block or genetic knockdown of ANO1 produces a similar inhi- bition of the contraction of mouse pulmonary artery to 5-HT as blocking VGCC or emptying Ca2+ stores from the SR. (A) Typical isometric force recordings in response to high K+ Krebs (85.4 mM) and increasing cumulative concen- trations of 5-HT ranging from 0.01 to 30 μM as indicated by the bars above the traces, in the absence (left) or presence (right) of the ANO1 inhibitor CaCCInh-A01, also indicated by a hori- zontal bar above the trace. (B) Mean cumulative dose–response curves to 5-HT in mouse pul- monary arteries from wild-type C57/BL6 mice in the absence (black circles, Control; n = 14), or presence (blue squares; n = 5) of 1 μM nifedipine to block VGCC, 10 μM CPA to deplete SR Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Blocking Assay, Knockdown, Control

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 2. The ANO1 blocker CaCCInh-A01 produced no effect on the high K+-mediated contraction of the mouse pulmonary artery. (A) Typical contractile force experiment showing that increasing the concentration of CaCCInh-A01 from 1 to 30 μM (progressively thickening black bar shown over the trace) produced no notice- able effect on the contraction (blue trace) eli- cited by 85.4 mM K+–Krebs solution (K+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Produced, Concentration Assay

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 3. Ca2+ oscillations triggered by 5-HT in individual smooth muscle cells from an intact mouse endothelium-denuded PA are potently inhibited by the inhibition of ANO1. All data were collected from the same PA from a conditional smooth muscle–specific and inducible GCaMP3 mouse injected with tamoxifen to induce Cre expression. (A) Ca2+ imaging was performed in the absence of an agonist (Control). The left panel shows one image from a video from which a ST map (middle colored image) was created in the area spanned by the diagonal white line. Fluorescence intensity was measured under the three white lines on the ST map (corresponding to two different cells) and plotted as a function of time as shown on the right. There was no detectable activity in these two cells as well as across the entire field of view of the movie. (B) Same nomenclature as in A except that the preparation was exposed to 1 μM 5-HT for 5 min. A ST map created in the same manner as that in A shows clear evidence of asynchronous Ca2+ transients. This is more evident from examining the fluorescence intensity profile of the same two cells analyzed in A, which displayed repetitive Ca2+ transient of distinct magnitude and frequency. (C) The nomenclature of this panel is identical to that of B and C, with the exception that the PA was exposed to 10 μM CaCCInh-A01 for 10 min while still being incubated with 5-HT. Examination of the ST map reveals little, if any, Ca2+ oscillations in the presence of the ANO1 inhibitor; Ca2+ transients were no longer apparent in the same two cells analyzed in A and B.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Inhibition, Injection, Expressing, Imaging, Control, Fluorescence, Activity Assay, Incubation

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 5. Sample experiment illustrating how ANO1 knockdown exerted a strong inhibition of 5-HT-induced Ca2+ oscillations in a PA from a tamoxifen-injected SMC-ANO1-KO-ΔEx12-GCaMP3 mouse. The top left panel is an image from a video stack recorded in a pulmonary artery from a con- ditional smooth muscle cell-specific and inducible ANO1 knockout mouse expressing GCaMP3 specifically in smooth muscle cells, which was exposed to 1 μM 5- HT for 5 min. One ST map constructed from the white line crossing the image is shown in the lower left corner and reveals very little activity. The fluorescence intensity profile as a function of time of two cells from the ST map labeled with the letters a and b are shown on the right. Cell 1 displayed no significant Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Knockdown, Inhibition, Injection, Knock-Out, Expressing, Construct, Activity Assay, Fluorescence, Labeling

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 6. Asynchronous Ca2+ oscillations evoked by 5-HT require both functional ANO1 and VGCC. Mean data for each of four parameters measured from Ca2+ transients elicited by 1 μM 5-HT (5 min) in PA from control SMC-GCaMP3 (light blue bars) or SMC-ANO1-KO-ΔEx12-GCaMP3 (light gray bars) mice. (A–D) The frequency of Ca2+ oscillations (A), peak Ca2+ transient amplitude (F/F0; B), integrated area under the curve (C), and FWHM (D) were measured as shown in the upper right corner. For each dataset, the mean is indicated by a filled black square with the colored boxes and whiskers delimiting the 25th and 75th percentile, and the 10th and 90th percentile of the pooled data, respectively, and small dots individual data points. N: number of animals; n: number of cells. SMC-GCaMP3 + 5-HT: N = 7, n = 114 for peak, area under the curve, and FWHM, and n = 116 for frequency; SMC-GCaMP3 + 5-HT + CaCCInh-A01 (CaCCInh): N = 7, n = 15 for peak, area under the curve, and FWHM, and n = 76 for frequency; SMC-GCaMP3 + 5-HT + nifedipine (Nif): N = 2, n = 32 for peak, area under the curve, and FWHM, and n = 47 for frequency; GCaMP3 + 5-HT + CPA: N = 2, n = 29; SMC-ANO1-KO-ΔEx12-GCaMP3; 5-HT: N = 7, n = 39 for peak, area under the curve, and FWHM, and n = 137 for frequency. For all panels, ***, **, and * indicate a significant difference between means with P < 0.001, P < 0.01, and P < 0.05, respectively.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Functional Assay, Control

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 7. Blocking ANO1 or CaV1.2 depletes SR Ca2+ stores. (A) Typical isometric force re- cording obtained under control conditions showing the effect of depleting the SR Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Blocking Assay, Control

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 8. ANO1, CaV1.2, and IP3R colocalize in peripheral coupling sites to form signaling complexes. (A and B) Co-IP of CaV1.2 or IP3R with ANO1 from lysates of the pulmonary artery from wild-type mice. Pulldown was carried out with anti-ANO1 antibody and then probed by Western blot with anti-CaV1.2, anti-IP3R, or anti- ANO1 antibodies. Five to six mouse tissues per experiment, each ran in triplicates. (C and D) Freshly isolated PASMCs from wild-type mice were immunolabeled for ANO1 and CaV1.2 (C) or ANO1 and IP3R (D). All three proteins were preferentially localized to the periphery of the cells. (D and F) Line profiles of the areas indi- cated by the white dashed lines in C and E. The fluorescence intensity was normalized to the minimum and maximum fluorescence for each sample. The black arrowheads denote the loca- tion of the PM. ANO1 and CaV1.2 show strong immunolabeling at the PM (D). (E) IP3R shows some intracellular immunolabeling, with moder- ate peaks present at the periphery showing an enhancement of protein localization to periphe- ral coupling sites. Source data are available for this figure: SourceData F8.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Isolation, Immunolabeling, Fluorescence

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 9. Superresolution imaging of ANO1, CaV1.2, and IP3R at the PM of PASMCs from wild-type mice. (A and B) Superresolution images of PASMCs labeled for ANO1 and CaV1.2 (A) or ANO1 and IP3R (B) were imaged using GSDIM in epifluorescence mode. Epifluorescence images are shown in the inset for

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Imaging, Labeling

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 10. Membrane cholesterol depletion with MβCD causes the internalization of ANO1 and CaV1.2 proteins. (A and C) Freshly isolated PASMCs from wild-type mice were im- munolabeled for ANO1 and CaV1.2 before (A) or after (C) a 30-min exposure to MβCD (3 mg/ml; MβCD) to deplete membrane cholesterol and disrupt lipid rafts. The two ion channel proteins were preferentially localized to the periphery of the cells in control conditions as similarly shown in Fig. 8. (B–D) Line profiles of the areas indi- cated by the white dashed lines in A and C are respectively displayed in B and D. For these plots, the fluorescence intensity was normalized to the minimum and maximum fluorescence for each sample. The black arrowheads denote the location of the PM. ANO1 and CaV1.2 show strong immunolabeling at the PM in control condition (C) and translocation toward the cen- ter core of the cell after exposure to MβCD (D). The cells from A and C were isolated from the same mouse. (E and F) Graphs summarizing the effects of exposing PASMCs to MβCD on the distribution of ANO1 (magenta bars) and CaV1.2 (green bars), respectively. Measurements were performed as described in the text and consisted in normalizing membrane fluorescence to total cell fluorescence. For each dataset, the mean is indicated by a large, filled black square with the colored boxes and whiskers delimiting the 25th and 75th percentile, and the 10th and 90th percentile of the pooled data, respectively, and small dots individual data points. N: number of animals; n: number of cells; for the control group (E): ANO1 and CaV1.2: N = 3, n = 43; for the MβCD group (F): ANO1 and CaV1.2: N = 3, n = 35. *** indicates a significant difference between means with P < 0.001.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Membrane, Isolation, Control, Fluorescence, Immunolabeling, Translocation Assay

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 12. Hypothetical models of EC coupling involving ANO1, CaV1.2, and IP3R during agonist-mediated contraction of mouse pulmonary ar- terial smooth muscle cells. (A) General uniform model depicting the acti- vation of ANO1 by both Ca2+ release from IP3-sensitive SR Ca2+ stores and Ca2+ entry through CaV1.2. In this model, the three ion transporters are evenly distributed in the membrane and are not physically coupled. The depolari- zation is maintained by the positive feedback loop established by CaV1.2- mediated activation of Cl−efflux through ANO1 and its impact on the state of activation of CaV1.2 through regulation of membrane potential. (B) Schematic diagram illustrating the local interaction of ANO1, CaV1.2 with IP3R and their impact on membrane potential, Ca2+ entry, and contraction. In this model, the three ion channels are physically coupled in a restricted number of sites (Super Cluster) distributed across the long axis of the cell (shown as red boxes in the bottom diagram) and are organized for compartmentalized Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Membrane, Activation Assay