Journal: American journal of physiology. Cell physiology

Article Title: Mouse organoid culture is a suitable model to study esophageal ion transport mechanisms.

doi: 10.1152/ajpcell.00295.2021

Figure Lengend Snippet: Figure 3. Expression of ion transporters in esophageal organoids (EOs). A: mature EOs were collected 9 days after plating, and RNA was prepared from the organoids. Gene expression of ion transporters was investigated with traditional RT-PCR analysis. B: immunostaining of EOs for Slc9a1 (first line), Slc9a2 (second line), Slc26a3 (third line), Slc4a4 (fourth line), and ANO1 (fifth line). The scale bar represents 100 mm (main photo) and 50 mm (inset photo), respectively. C: costaining of Slc26a6 (red) and cftr (green). The scale bar represents 50 mm (upper line), 25 mm (middle line), and 10 mm (bottom line), for both mice strains. CFTR, cystic fibrosis transmembrane conductance regulator.

Article Snippet: List of primary and secondary antibodies used in the study Primary Antibodies Company Cat. No. Dilution Slc9a1 Alomone ANX-010 1:100 Slc9a2 Alomone ANX-002 1:100 Slc26a3 Invitrogen PA5-68530 1:600 Slc26a6 Santa Cruz Biotechnology sc-515230 1:200 Slc4a4 Abcam ab187511 1:100 CFTR Alomone ACL-006 1:200 ANO1 Alomone ACL-011 1:200 CFTR, cystic fibrosis transmembrane conductance regulator.

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Immunostaining

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 1. ANO1 is upregulated in metastatic tumors and correlated with poor prognosis in ESCC. A, Flow chart of the strategy for the screening of cancer metastasis drivers. B, Heatmap demonstrating the differentially expressed genes between metastatic tissues and primary ESCC tumors. C, Venn diagram was used to overlap the genes upregulated in ESCC lymph node metastasis tissues and highly metastatic ESCC cell subline. D and E, The expression of ANO1 was examined in three cases of primary ESCC tumors and paired lymph node metastasis tissues by qRT-PCR (D) and Western blot analysis (E). F, Representative immunohistochemical images and quantitative analysis of ANO1 staining in 100 ESCC tissues and 75 matched normal tissues. G, Survival analysis of 100 patients with ESCC stratified by the ANO1 level. H, Representative images and quantitative analysis of ANO1 staining in 40 ESCC tissues and the matched metastatic samples. I, The expression level of ANO1 in the cohort of esophageal carcinoma (ESCA) in the TCGA database. J, Analysis of the ANO1 expression in esophageal cancer patients with different nodal metastasis status on UALCAN website. K, Analysis of the ANO1 level in patients with HNSC, KIRC, PCPG, and STAD in TCGA database. Bars, SD. , P < 0.05; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 2. ANO1 promotes cancer metastasis in vitro and in vivo. A and B, The invasive ability of ANO1-overexpressing (A) or ANO1-knockdown (B) ESCC cells was examined by Boyden chamber invasion assay. C and D, The protein expression of b-catenin, E-cadherin, and snail was compared in ANO1-overexpressing (C) or ANO1-knockdown (D) ESCC cells by Western blot analysis. E, Representative images of swollen inguinal lymph nodes and primary tumors in mice receiving subcutaneous footpad injection of ANO1-overexpressing ESCC cells or control cells (6 mice per group). F, Hematoxylin and eosin staining of invaded tumor in lymph node metastasis model. G, Mice were intravenously injected with ANO1-overexpressing or control cells and the lung colonization was detected using bioluminescence imaging (6 mice per group). H, Hematoxylin and eosin staining of lung sections in lung colonization model. I–L, The effect of ANO1 knockdown on the metastasis of ESCC cells to the lymph nodes and lungs. Bars, SD. , P < 0.01; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: In Vitro, In Vivo, Knockdown, Invasion Assay, Expressing, Western Blot, Injection, Control, Staining, Imaging

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 3. ANO1 increases intracellular cholesterol level via inactivation of LXR pathway. A, Volcano plot of differentially expressed genes (fold change > 2; P < 0.05) in ANO1-overexpressing ESCC cells by RNA-seq. B, Gene ontology analysis indicated the alternation of LXR signaling in ANO1-overexpressing cells. C and D, The mRNA and protein levels of ABCA1 and ABCG1 in ANO1-overexpressing ESCC cells were examined by qRT-PCR and Western blot analysis. E and F, Effect of ANO1 knockdown on ABCA1 and ABCG1 expression in ESCC cells. G and H, Intracellular cholesterol level in ESCC cells with manipulation of ANO1 expression. I, Intracellular cholesterol level of ANO1-overexpressing ESCC cells in presence or absence of GW3965 (5 mmol/L). J, Relative mRNA levels of ABCA1 and ABCG1 in ANO1-overexpressing ESCC cells in presence or absence of GW3965 (5 mmol/L). K, The invasive abilities of ANO1-overexpressing ESCC cells and control cells were determined in the presence or absence of GW3965 (5 mmol/L) or M-b-CD (2.5 mmol/L). L and M, Comparison of the lung colonization of ANO1-overexpressing ESCC cells and control cells in the mice with or without GW3965 (10 mg/kg) or M-b-CD (64 mg/kg) treatment. Bars, SD. , P < 0.01; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Knockdown, Expressing, Control, Comparison

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 4. ANO1 interacts with JUN to inhibit CYP27A1 transcription and to repress cholesterol hydroxylation. A and B, qRT-PCR and Western blot analysis of the expression of CYP27A1 in ANO1-overexpressing ESCC and control cells. C and D, Effect of ANO1 knockdown on CYP27A1 expression in ESCC cells. E, The luciferase activity of CYP27A1 promoter was determined in the ESCC cells with overexpression of ANO1. (Continued on the following page.)

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control, Knockdown, Luciferase, Activity Assay, Over Expression

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 5. ANO1-expressing ESCC cells induce IL1b secretion and activate fibroblasts. A, Diagram showing the coculture systemof ESCC cells and fibroblasts. B, The migration of fibroblasts attracted by the CM from ANO1-overexpressing ESCC cells and control cells was determined by Boyden chamber assay. C, The expression of aSMA, FAP in the fibroblasts treated with indicated CM was examined by Western blot analysis. D, Heatmap of the differentially expressed proteins in the CM of ANO1- overexpressing ESCC cells detected by cytokine array assay. E and F, The expression and secretion levels of IL1b were determined by Western blot analysis (E) and ELISA (F) in ANO1-overexpressing ESCC cells and control cells. G and H, Knockdown of ANO1 in ESCC cells decreased IL1b expression and secretion. I and J, Effect of ANO1 manipulation on IL1b mRNA level in ESCC cells. K and L, The mRNA and protein expression of IL1b was determined in ANO1-overexpressing cells with or without GW3965 (5 mmol/L) treatment. M, Further knockdown of IL1b in ESCC cells or the addition of IL1b neutralizing antibody reversed the migration of fibroblasts induced by ANO1-overexpressing ESCC cells. N, Fibroblast activation markers aSMA and FAP were detected by Western blot analysis in the fibroblasts treated with the indicated CM. Bars, SD. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Expressing, Migration, Control, Boyden Chamber Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Activation Assay

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 6. IL1b-activated fibroblasts induces CCL1 secretion to exert positive feedback on ESCC cancer cell invasion. A, Flow chart showing the collection of CM from activated fibroblasts to attract the invasion of ESCC cells. B, Invasion of KYSE150 and EC9706 cells exposed to the different fibroblast-derived CM. C, Radar map illustrating the top 30 upregulated cytokines in the CM of fibroblasts treated with supernatant of ANO1-overexpresing ESCC cell by cytokine antibody array. D, After transfection with the siRNAs targeting the top 10 upregulated cytokines, respectively, the fibroblasts were treated with rIL1b (20 ng/mL) and the CM were collected to attract the invasion of KYSE150 cells. E, The secretion of CCL1 from the fibroblasts exposed to rIL1b (20 ng/mL) for 48 hours was determined by ELISA assay. F, Western blot was used to detect p-p65 and p65 expressionin the fibroblasts stimulated with rIL1b (20 ng/mL) in presence or absence of CAPE (10 mmol/L). G andH, qRT-PCR and ELISA analyses of CCL1 expression and secretion in the fibroblasts exposed to rIL1b (20 ng/mL) with or without addition of CAPE (10 mmol/L). I, Invasion of KYSE150 and EC9706 cells exposed to the different fibroblast-derived CM in presence or absence of CAPE. Bars, SD. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Derivative Assay, Ab Array, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Expressing

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 7. Lead compound K786–4469 targets ANO1 to suppress tumor metastasis. A, Schematic diagram of screening strategies for the ANO1-targeting small molecule compounds. B, The inhibitory effects of 24 candidate compounds on ESCC cell invasion were compared using Boyden chamber assay. C, The expression of ANO1, CYP27A1, ABCA1, and ABCG1 in ESCC cells treated with increasing concentrations of K786–4469 (up to 10 mmol/L) was detected by Western blot. D, Effect of K786– 4469 on mRNA levels of ABCA1, ABCG1, and CYP27A1. E, The intracellular cholesterol level was determined in K786–4469-treated ESCC cells. F, K786–4469 repressed ESCC cell invasion in a dose-dependent manner. G, Mice were intravenously injected with KYSE150-Luc-LM3 cells and treated with K786–4469 or DMSO; lung colonization was detected using bioluminescence imaging. H, Hematoxylin and eosin staining of lung sections as indicated. I, The structure of the ANO1 protein complexed with K786–4469. J, Wild-type or different mutant ANO1 was re-overexpressed in ANO1-knockdown ESCC cells, and suppressive effects of K786–4469 were compared by using Boyden chamber assay. K, Lung colonization in the mice injected with the indicated cell lines and treated with K786–4469 or DMSO was detected using bioluminescence imaging. Bars, SD. n.s., nonsignificant; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Boyden Chamber Assay, Expressing, Western Blot, Injection, Imaging, Staining, Mutagenesis, Knockdown

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 8. Working model of ANO1 promotes cancer metastasis by regulating CYP27A1–LXR signaling. ANO1 interacts with JUN to inhibit the transcription of CYP27A1 and inactivates LXR signaling, leading to increased cholesterol level and microenvironment reprogramming, therefore promoting cancer metastasis in ESCC. [The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license (https://smart.servier.com).]

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Generated

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: Expression of TMEM16A in hepatocellular carcinoma and pericarcinous tissue. Notes: Expression of TMEM16A at ( A ) mRNA level and ( B ) protein level. ( C ) Quantification of the protein bands (OD ratio over GAPDH). Tumor: hepatocellular carcinoma; Normal: pericarcinous tissue. ** P <0.01. Abbreviations: OD, optical density; N, normal tissues; T, tumor tissues.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: Expression of TMEM16A in SMMC-7721 cells after TMEM16A siRNA transfection. Notes: ( A ) qRT-PCR analyzed the mRNA expression of TMEM16A in SMMC-7721 cells after TMEM16A-siRNA transfection. ( B ) The TMEM16A protein expression level in SMMC-7721 cells after TMEM16A-siRNA transfection was detected using Western blot. Control: normal SMMC-7721 cells; siRNA: cells transfected with TMEM16A siRNA. ** P <0.01 vs control. Abbreviation: NC, negative control.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Control, Negative Control

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: The proliferation, migration, and invasion of SMMC-7721 cells were attenuated by knockdown of TMEM16A. Notes: ( A ) After transfection, the cell proliferation was detected by MTT assay. ( B ) The migration and invasion were assessed by transwell chamber, and ( C ) migrated cells were counted. Control: normal SMMC-7721 cells; NC: negative control; siRNA: cells transfected with TMEM16A siRNA. * P <0.05 and ** P <0.01 vs control. Abbreviation: h, hours.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Migration, Knockdown, Transfection, MTT Assay, Control, Negative Control

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: Cell cycle and cell apoptosis of SMMC-7721 cells that were transfected with TMEM16A siRNA. Notes: ( A ) Cell cycle distribution was measured by flow cytometry with cell cycle staining kit and ( B ) the cell cycle phase is shown in a bar graph with the G0/G1, S, and G2/M phases. ( C ) Cell apoptosis in each group was determined using the Annexin V-FITC/PI flow cytometry, and ( D ) proportion of apoptosis cells was measured. Control: normal SMMC-7721 cells; NC: negative control; siRNA: cells transfected with TMEM16A siRNA. * P <0.05 vs control. Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide; V-FITC/PI, fluorescein isothiocyanate-conjugated Annexin V and PI.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Transfection, Flow Cytometry, Staining, Control, Negative Control

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: Expression of MAPK signaling proteins (p38, p-p38, ERK1/2, p-ERK1/2, JNK, and p-JNK) and cell cycle regulatory protein cyclin D1 in TMEM16A siRNA-transfected SMMC-7721 cells. Abbreviation: NC, negative control.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Expressing, Transfection, Negative Control

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: Knockdown of TMEM16A suppresses tumorigenicity in vivo. Notes: Tumor volume ( V ) was measured daily by caliper and was calculated using the formula V = ( L × W 2 )/2, where L was the length and W was the width of the tumor. Growth curves were plotted using average tumor volume within each experimental group every week after inoculation of TMEM16A shRNA-transfected SMMC-7721 cells and NC shRNA-transfected SMMC-7721 cells. Six weeks later, the mice were euthanized, and ( A ) the dissected tumors were collected and measured (in cm), and ( B ) the tumor growth curves were determined. * P <0.05 siRNA vs NC. Abbreviation: NC, negative control.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Knockdown, In Vivo, shRNA, Transfection, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Endogenous Ca 2+ -dependent TMEM16A and cAMP-activated CFTR Cl − transport in HT 29 colonic epithelial cells. ( A , B ) YFP fluorescence quenching by iodide is enhanced by ATP in a concentration-dependent manner ( n = 5 for all). siRNA knockdown of TMEM16A but not TMEM16F inhibits quenching. ( C ) Semiquantitative RT-PCR indicates knockdown of TMEM16A (T16A; 92%, n = 3) and TMEM16F (T16F; 93%, n = 3). ( D , E ) Western blotting indicating pronounced expression of endogenous TMEM16A and CFTR in HT 29 cells. siRNA knocked down expression of TMEM16A. ( F – H ) Activation of chloride conductance by IBMX and forskolin (I/F; 100 µM/2 µM) was not detected in iodide quenching ( n = 5), but was significant in whole cell patch clamp recordings (overlay currents and I/V curves; n = 5). Mean ± SEM. * significant activation ( p < 0.05; paired t -test). # significant inhibition ( p < 0.05; ANOVA).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Fluorescence, Concentration Assay, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Activation Assay, Patch Clamp, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Eact does not activate endogenous Ca 2+ -dependent Cl − secretion, and has little effect on [Ca 2+ ] i . ( A , B ) No activation of iodide quenching by Eact or GSK1016790 ( n = 5 for both). ( C ) RT-PCR indicating the expression of TRPV4 in HT 29 cells. ( D , E ) No activation of whole cell currents in HT 29 cells by Eact or GSK1016790 (10 µM; n = 5 for both). ( F , G ) RT-PCR of TRPV4 expressed in CFBE ( F ) and HEK293 ( G ) cells. ( H ) ATP (100 µM) induced [Ca 2+ ] i rise is inhibited by knockdown of TMEM16A but not by Ani9 (10 µM; n = 37–105). ( I , J ) Minor increase in [Ca 2+ ] i by Eact in HT 29 , CFBE, or HEK293 cells ( n = 31–111). Mean ± SEM. # significant inhibition ( p < 0.05; unpaired t -test).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Knockdown, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Effect of inhibitors on Ca 2+ -activated Cl − transport and ATP-induced rise in [Ca 2+ ] i . A ) Increase in intracellular Ca 2+ by stimulation of HT 29 cells with ATP (100 µM). In contrast to benzbromarone and niclosamide, Ani9 did not attenuate the effect of ATP on [Ca 2+ ] i ( n = 7–23). In Call33 head and neck cancer cells and M1 mouse collecting duct cells Ani9 (10 µM) inhibited ATP-induced Ca 2+ increase ( n = 134–162) significantly. B ) Inhibition of ATP (5 µM) activated YFP-quenching in HT 29 cells by BBR (10 µM). C ) Inhibition of ionomycin (Iono, 1 µM) activated whole cell currents by BBR in HEK293 cells overexpressing TMEM16A (original recording and I/V curves) ( n = 5–6). D ) Inhibition of ionomycin (Iono; 1 µM) activated whole cell currents by BBR in HEK293 cells overexpressing TMEM16F ( n = 6, original recording and I/V curves). E ) Inhibition of ATP (5 µM) induced YFP-quenching in HT 29 cells by Ani9. F , G ) Inhibition of ionomycin (Iono; 1 µM) activated TMEM16A currents by Ani9 (10 µM), and change in time-dependent activation of TMEM16F currents ( n = 5–8). * significant inhibition ( p < 0.05; paired t -test). # significant inhibition ( p < 0.05; unpaired t -test).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Inhibition, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Niclosamide inhibits TMEM16A currents activated by intracellular Ca 2+ . (A ) TMEM16A overexpressed in HEK293 cells was activated by 1 µM Ca 2+ in the patch pipette filling solution. Acute application of niclosamide (Niclo; 5 µM) significantly inhibited Ca 2+ -activated TMEM16A whole cell currents ( n = 7). ( B ) 15 min preincubation with Niclo inhibited TMEM16A more potently ( n = 9–10). ( C ) Increase in intracellular Ca 2+ with ATP (100 µM) in HT 29 cells. Niclosamide (5 µM) induces a slight and transient increase in [Ca 2+ ] i and inhibits ATP-induced rise in [Ca 2+ ] i . siRNA knockdown of TMEM16A inhibits increase in [Ca 2+ ] i by ATP. Niclosamide shows no additional effects on [Ca 2+ ] i ( n = 60–193). ( D ) ER Ca 2+ store release and Ca 2+ influx (SOCE) induced by CPA and niclosamide ( n = 40–50). ( E , F ) Effects of CPA and niclosamide on Ca 2+ store release and SOCE under various conditions ( n = 40–213) * significant inhibition ( p < 0.05; paired t -test). # significant inhibition ( p < 0.05; unpaired t -test).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Transferring, Knockdown, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Endogenous and overexpressed TMEM16A behave differently. ( A ) Activation of overexpressed TMEM16A whole cell currents in HEK293 cells by Eact (10 µM, n = 9). ( B ) Activation of TMEM16F whole cell currents in TMEM16F-overexpressing HEK293 cells by Eact (10 µM, n = 6). ( C , D ) Little activation of endogenous TMEM16A currents by melittin (200 nM; n = 10) in HT29 cells, but strong activation in HEK293 cells overexpressing TMEM16A ( n = 7). ( E , F ) Little activation of endogenous TMEM16A currents by cinnamaldehyde (Cinna; 1 µM; n = 9), but strong activation of overexpressed TMEM16A ( n = 5). ( G , H ) Little activation of endogenous TMEM16A currents by diC8-PIP 2 (50 µM; n = 6), but strong activation of overexpressed TMEM16A ( n = 6). * significant activation ( p < 0.05; paired t -test). # significant activation ( p < 0.05; unpaired t -test).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Effects of potential activators/potentiators of TMEM16A in HT29 cells (endogenous TMEM16A) and HEK293 cells (overexpressed TMEM16A).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: diC8-PIP2 augments TMEM16A currents activated by ionomycin. ( A – D ) Whole cell currents and I/V curves showing effect of diC8-PIP 2 (50 µM in the patch pipette filling solution) on basal and ionomycin (Iono, 0.1 µM) activated TMEM16A currents in HT 29 cells ( A , B ) and HEK293 cells ( C , D ). Activation of TMEM16A by diC8-PIP2 is clearly observed in TMEM16A-overexpressing HEK293 cells but not in HT 29 cells ( n = 6–7 for all). ( E , F ) Time courses for Iono-activated TMEM16A currents in HT 29 and HEK293 cells ( n = 6–8). * significant activation ( p < 0.05; paired t -test). # significant difference to the absence of diC8-PIP 2 ( p < 0.05; unpaired t -test).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Transferring, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: RT-PCR primers.

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques:

Journal: Molecular medicine reports

Article Title: TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway.

doi: 10.3892/mmr.2016.4979

Figure Lengend Snippet: Figure 2. Decreased expression of TMEM16A, p‑MLC/MLC and p‑MYPT1/MYPT1 in rat basilar arteries during the development of hypertension. (A) Western blot analysis of TMEM16A in basilar arteries at various time‑points after 2‑kidney, 2‑clip surgery. Western blot analysis of phosphorylation of (B) MLC and (C) MYPT1 in basilar arteries. GAPDH served as a loading control. *P<0.05, **P<0.01 vs. week 0 (n=6). TMEM16A, transmembrane protein 16A; MLC, myosin light chain; MYPT1, myosin phosphatase‑targeting subunit 1; p, phosphorylated.

Article Snippet: Rabbit polyclonal TMEM16A antibody was obtained from Novus Biologicals, LLC (Littleton, CO, USA; cat. no. NBP1‐60076; dilution, 1:500).

Techniques: Expressing, Western Blot, Phospho-proteomics, Control

Journal: Molecular medicine reports

Article Title: TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway.

doi: 10.3892/mmr.2016.4979

Figure Lengend Snippet: Figure 3. Ang II evoked a TMEM16A‑mediated current using 100 nM [Ca2+]i in BASMCs. (A) Representative traces of ICl.Ca recorded in BASMCs. Cells were immersed in a bath solution and the current was recorded using a whole‑cell patch clamp with (a) a basal intracellular calcium concentration (100 nM [Ca2+]i) and (b) immediately after 100 nM Ang II was perfused into the bath solution. Results from BASMCs pretreated with (c) TMEM16A siRNA and 100 nM [Ca2+]i, 100 nM Ang II‑induced current for 48 h or (d) 10 µM losartan for 15 min. (e) Bar graph of current density at 100 mV in the different groups. (B) IClCa was recorded in BASMCs with (a) 500 nM [Ca2+]i and (b) the current wasn't enhanced by 100 nM Ang II. (c) Bar graph of current density at 100 mV in the two groups. *P<0.05, **P<0.01 (n=8‑11). Ang II, angiotensin II; TMEM16A, transmembrane protein 16A; [Ca2+]i, intracellular Ca2+; BASMC, basilar artery smooth muscle cell; IClCa, Ca2+‑dependent Cl− channel.

Article Snippet: Rabbit polyclonal TMEM16A antibody was obtained from Novus Biologicals, LLC (Littleton, CO, USA; cat. no. NBP1‐60076; dilution, 1:500).

Techniques: Patch Clamp, Concentration Assay

Journal: Molecular medicine reports

Article Title: TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway.

doi: 10.3892/mmr.2016.4979

Figure Lengend Snippet: Figure 4. Effect of si‑TMEM16A and adenovirus on TMEM16A expression in BASMCs. BASMCs were treated with (A) si‑TMEM16A or (B) adv‑TMEM16A for 3 and 6 h, respectively, and then cultured with 10% fetal bovine serum and Dulbecco's modified Eagle's medium/Ham's F-12 medium for another 48 h. TMEM16A protein expression was detected by western blot analysis and GAPDH served as a loading control. *P<0.05 vs. Neg Ctrl. n=4. TMEM16A, transmembrane protein 16A; BASMC, basilar artery smooth muscle cell; Neg Ctrl, negative control; si‑TMEM16A, TMEM16A siRNA; adv‑TMEM16A, TMEM16A adenovirus.

Article Snippet: Rabbit polyclonal TMEM16A antibody was obtained from Novus Biologicals, LLC (Littleton, CO, USA; cat. no. NBP1‐60076; dilution, 1:500).

Techniques: Expressing, Cell Culture, Modification, Western Blot, Control, Negative Control

Journal: Molecular medicine reports

Article Title: TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway.

doi: 10.3892/mmr.2016.4979

Figure Lengend Snippet: Figure 5. Regulation of Ang II‑induced phosphorylation of MLC and MYPT1 by TMEM16A expression. BASMCs were treated with (A and B) si‑TMEM16A or (C and D) adv‑TMEM16A for 3 h or 6 h and cultured for another 48 h. Y‑27632 (10 µM) was added 10 min before the 5‑min treatment of 100 nM Ang II. Cell lysates were collected and phosphorylation of (A and C) MLC and (B and D) MYPT1 were detected by western blot analysis. GAPDH served as a loading control. *P<0.05; n=8. Ang II, angiotensin II; MLC, myosin light chain; MYPT1, myosin phosphatase‑targeting subunit 1; TMEM16A, transmembrane pro tein 16A; si, small interfering; si‑TMEM16A, TMEM16A siRNA; BASMC, basilar artery smooth muscle cell; p, phosphorylated; adv‑TMEM16A, TMEM16A adenovirus.

Article Snippet: Rabbit polyclonal TMEM16A antibody was obtained from Novus Biologicals, LLC (Littleton, CO, USA; cat. no. NBP1‐60076; dilution, 1:500).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Western Blot, Control

Journal: Molecular medicine reports

Article Title: TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway.

doi: 10.3892/mmr.2016.4979

Figure Lengend Snippet: Figure 6. Expression of GTP‑RhoA/RhoA in cultured BASMCs in response to 100 nM Ang II following TMEM16A downregulation or overexpression. BASMCs were treated with (A) si‑TMEM16A or (B) adv‑TMEM16A for 3 h or 6 h and then cultured with 10% fetal bovine serum and Dulbecco's modified Eagle's medium/Ham's F-12 medium for another 48 h. AngII (100 nM) was added for 5 min to induce cell contraction. Cell lysates were collected and GTP‑RhoA and RhoA were detected by western blot analysis. GAPDH served as a loading control. *P<0.05; n=8. GTP, guanosine-5'-triphosphate; BASMC, basilar artery smooth muscle cell; TMEM16A, transmembrane protein 16A; si, small interfering; si‑TMEM16A, TMEM16A siRNA; adv‑TMEM16A, TMEM16A adenovirus; Neg Ctrl, negative control.

Article Snippet: Rabbit polyclonal TMEM16A antibody was obtained from Novus Biologicals, LLC (Littleton, CO, USA; cat. no. NBP1‐60076; dilution, 1:500).

Techniques: Expressing, Cell Culture, Over Expression, Modification, Western Blot, Control, Negative Control

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: KN-93 reduces the surface expression and activity of ANO1 in U251 cells. ( A ) U251 cells treated with DMSO or KN-93 were imaged using antibodies against ANO1 and WGA647 (WGA), a plasma membrane marker. Scale bar, 20 μm. ( B ) The Pearson’s correlation coefficient for ANO1 with KN-93 was significantly less than the value obtained for ANO1 with DMSO in U251 cells. ( C ) Cell surface biotinylation results from membrane protein fractions from U251 cells treated with DMSO or KN-93. ( D ) The summary bar graph showing data obtained from three independent experiments as in ( C ). ( E ) Averaged traces of whole-cell currents of U251 cells treated with DMSO or T16Ainh-A01, an ANO1 inhibitor. ( F ) The summary bar graph shows the inhibitory effect of KN93 or T16Ainh-A01 on ANO1 current amplitude at ±100 mV. ( G ) Averaged traces of normalized T16Ainh-A01-sensitive currents of U251 cells treated with DMSO or KN-93. ( H ) The bar graph shows normalized T16Ainh-A01-sensitive current densities ( G ) at + 100 mV. Number on each bar indicates n for each condition. All values are mean ± s.e.m. P -values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001. n.s means not significant.

Article Snippet: To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene (Rockville, Maryland, USA, TL300993), which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D).

Techniques: Expressing, Activity Assay, Clinical Proteomics, Membrane, Marker

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: CaMKIIβ specifically increases the surface expression and activity of ANO1 in U251 cells. ( A ) The mRNA expression of CaMKII isoforms in U251 cells were detected with specific primers for CaMKIIα, β, γ and δ. ( B ) Normalized expression of CaMKII isoforms as compared to that of GAPDH in U251 cells. ( C ) Effect of CaMKII isoform overexpression on ANO1-mediated whole cell chloride currents in U251 cells. ( D ) The summary bar graph showing ANO1-mediated current density at ± 100 mV. ( E ) Cell surface biotinylation results from membrane protein fractions from U251 cells transfected with mCh-CaMKIIα and mCh-CaMKIIβ. ( F ) The summary bar graph showing data obtained from at least three independent experiments as in ( E ). Number on each bar indicates n for each condition. All values are mean ± s.e.m. P -values were obtained with Student’s t-test. ** p < 0.01 and *** p < 0.001. n.s means not significant.

Article Snippet: To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene (Rockville, Maryland, USA, TL300993), which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D).

Techniques: Expressing, Activity Assay, Over Expression, Membrane, Transfection

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: CaMKIIβ knockdown reduces the surface expression of ANO1 in U251 cells. ( A,B ) Validation of the silencing efficiency of the siRNA against CaMKIIβ using qPCR and Western blotting. ( C ) U251 cells transfected with Sc shRNA or CaMKIIβ siRNA were imaged using antibodies against ANO1 and WGA. Nuclei were stained using DAPI staining solution. Scale bar, 20 μm. ( D ) Cell surface biotinylation results from U251 cells transfected with Sc shRNA or CaMKIIβ siRNA. ( E ) The summary bar graph shows the summary of ( D ), data obtained from three independent experiments. ( F ) Averaged traces of whole-cell currents of U251 cells transfected with Sc shRNA, CaMKIIβ siRNA, or T16Ainh-A01 (A01). ( G ) The summary bar graph shows the inhibitory effect of CaMKIIβ siRNA on ANO1–mediated current amplitude at ±100 mV. The bar graph shows normalized A01-sensitive current densities at + 100 mV. Number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with Student’s t-test. * p < 0.05 and ** p < 0.01.

Article Snippet: To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene (Rockville, Maryland, USA, TL300993), which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D).

Techniques: Knockdown, Expressing, Biomarker Discovery, Western Blot, Transfection, shRNA, Staining

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: Gene silencing of ANO1 or CaMKIIβ leads to the attenuation of migration and invasion in U251 cells. ( A,B ) Validation of the silencing efficiency of the shRNA against ANO1 using qPCR and Western blotting. ( C ) Cell invasion assay of U251 cells infected with Lenti-ANO1 shRNA and transfected with CaMKIIβ siRNA. ( D ) The averaged bar graph shows the summary of ( C ), data obtained from three independent experiments. ( E ) Representative photographic images of the migration of U251 cells infected with Lenti-ANO1 shRNA and transfected with CaMKIIβ siRNA and the cells with Lenti-Sc shRNA and Sc siRNA control. Scale bar, 100 μm. ( F ) The averaged bar graph shows the summary of ( E ), data obtained from three independent experiments. All values are mean ± s.e.m. p -values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene (Rockville, Maryland, USA, TL300993), which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D).

Techniques: Migration, Biomarker Discovery, shRNA, Western Blot, Invasion Assay, Infection, Transfection, Control

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: Inhibition of CaMKIIβ and/or ANO1 decreased migration and invasion of U87MG cells. ( A,B ) Validation of the silencing efficiency of the Lenti-ANO1shRNA and the CaMKIIβ siRNA with qPCR and Western blot in U87MG cells. ( C ) Cell invasion assay of U87MG cells infected with Lenti-ANO1shRNA and transfected with CaMKIIβ siRNA. ( D ) The averaged bar graph shows the summary of (C), data obtained from three independent experiments. ( E ) Representative photographic images of the migration of U87MG cells infected with Lenti-ANO1shRNA and transfected with CaMKIIβ siRNA as compared to the cells with Lenti-Sc shRNA and Sc siRNA control. Scale bar, 100 μm. ( F ) The averaged bar graph shows the summary of ( E ), data obtained from three independent experiments. All values are mean ± s.e.m. p -values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene (Rockville, Maryland, USA, TL300993), which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D).

Techniques: Inhibition, Migration, Biomarker Discovery, Western Blot, Invasion Assay, Infection, Transfection, shRNA, Control

Journal: Biochemistry and Biophysics Reports

Article Title: Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

doi: 10.1016/j.bbrep.2021.100912

Figure Lengend Snippet: Ano1 mRNA expressed differentially and carbachol (CCH) stimulates Isc both in mouse small and large intestine. (A) Represents the quantity of Ano1 gene expression. Data are means ± S.E (n = 3). (B) CCH stimulated Isc in different parts of the mouse intestine, as indicated. *P < 0.01 (ANOVA with Bonferroni's test). (C) Effects of MONNA on basal and CCH-stimulated Isc and (D) representative tracing of apical and serosal incubation of MONNA in response to CCH stimulation in mouse colonic tissue. Data are means ± S.E (n = 4–7). (E) Showing representative Isc response to CCH in the presence of apical to serosal and serosal to apical Cl − gradient. Inset indicates the direction of the Cl − gradient. Ap, apical and Bl, serosal side of the mouse colon (n = 3).

Article Snippet: Mouse intestinal tissue sections were fixed in 3% paraformaldehyde prior to paraffin embedding [ , ].Thereafter, sections were incubated with rabbit anti -TMEM16A (Novus biological, #NBP2-29662) and mouse anti -NHERF1(Santa Cruz Biotechnology, #sc-271552) antibody overnight at 4 °C followed by exposure to goat anti-rabbit- IgG Alexa Fluor 488 or goat anti-mouse-IgG Alexa fluor 680 secondary antibody (Invitrogen; 1:500) for 1h at room temperature.

Techniques: Gene Expression, Incubation

Journal: Biochemistry and Biophysics Reports

Article Title: Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

doi: 10.1016/j.bbrep.2021.100912

Figure Lengend Snippet: Ano1 ± mouse caused reduction of basal and CCH-stimulated Isc. (A) Basal and CCH-stimulated Isc in the small intestine (jejunal) and colonic tissue of WT and Ano1 ± mice. Data are means ± S.E (n = 6).*P < 0.05. (B) Showing FSK-stimulated Isc response in WT and Ano1 ± mouse colonic mucosa. Data are means ± S.E (n = 4–6). (C) Representative western blot image of TMEM16A protein in different parts of wild type and Ano1 ± enterocytes. (D) Quantitative analysis of the immunoblots was determined by ImageJ analysis. Data are means ± S.E of three independent experiments. (* denotes significant difference at p value < 0.05, NS, non significant at p < 0.05).

Article Snippet: Mouse intestinal tissue sections were fixed in 3% paraformaldehyde prior to paraffin embedding [ , ].Thereafter, sections were incubated with rabbit anti -TMEM16A (Novus biological, #NBP2-29662) and mouse anti -NHERF1(Santa Cruz Biotechnology, #sc-271552) antibody overnight at 4 °C followed by exposure to goat anti-rabbit- IgG Alexa Fluor 488 or goat anti-mouse-IgG Alexa fluor 680 secondary antibody (Invitrogen; 1:500) for 1h at room temperature.

Techniques: Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

doi: 10.1016/j.bbrep.2021.100912

Figure Lengend Snippet: TMEM16A localizes to the apical membrane of mouse enterocytes and immunoprecipitates with NHERF1 in co-transfected HEK293T cells. (A) Representative western blot showing NHERF1 expression in mouse intestinal tissue as indicated. The histogram showing the densitometric quantification of NHERF1 protein expression, right to the immunoblot. This experiment was performed in triplicate. (B) Immunofluorescence demonstrated TMEM16A (green) and NHERF1 (red) in mouse colon, the yellow signal indicated co-localization of TMEM16A with NHERF1 (merged). Representative of two independent experiments. (C) TMEM16A co-immunoprecipitates with Flag-NHERF1 in co-transfected HEK293T cells. TMEM16A was detected only in the Co-IP complex from cell co-transfected with plasmids expressing both mouse Ano1 and Flag-Nherf1. (D) Depicted a reduced TMEM16A abundance in Co-IP complex from cell lysates with truncated (delC4) Ano1 compared to the result obtained with the control (full length). Representative blot of three independent experiments for (C) and (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Mouse intestinal tissue sections were fixed in 3% paraformaldehyde prior to paraffin embedding [ , ].Thereafter, sections were incubated with rabbit anti -TMEM16A (Novus biological, #NBP2-29662) and mouse anti -NHERF1(Santa Cruz Biotechnology, #sc-271552) antibody overnight at 4 °C followed by exposure to goat anti-rabbit- IgG Alexa Fluor 488 or goat anti-mouse-IgG Alexa fluor 680 secondary antibody (Invitrogen; 1:500) for 1h at room temperature.

Techniques: Membrane, Transfection, Western Blot, Expressing, Immunofluorescence, Co-Immunoprecipitation Assay, Control

Journal: Biochemistry and Biophysics Reports

Article Title: Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

doi: 10.1016/j.bbrep.2021.100912

Figure Lengend Snippet: Functional involvement of NHERF1 in TMEM16A mediated Cl − secretion. (A) NHERF1 was co-immunoprecipitated with TMEM16A endogenously from T84 cell lysates. No signal was detected using protein-G-conjugated beads alone (IP control). Representation of three independent experiments. ( B ) Western blot showing NHERF1 expression in wild type T84 (T84WT), vector control (T84VC) and NHERF1 knocked down cells (T84NF1KD736). (C) Histogram showing densitometric quantification of NHERF1 expression, right to the immunoblot. Result represents means ± S.E (n = 3). (D ) Representative traces showing the response of T84NF1KD736 cells to CCH-stimulated Isc. Inset showing summarized data from six independent experiments. Data are means ± S.E. (E) Effect of FSK pre-stimulation on CCH-stimulated Isc in WT and NHERF1KD T84 cell monolayers. Monolayers grown on filters were exposed to FSK (10 μM) and then stimulated with serosal CCH (100 μM). Data are mean ± SEM from 4 to 6 monolayers of each condition.

Article Snippet: Mouse intestinal tissue sections were fixed in 3% paraformaldehyde prior to paraffin embedding [ , ].Thereafter, sections were incubated with rabbit anti -TMEM16A (Novus biological, #NBP2-29662) and mouse anti -NHERF1(Santa Cruz Biotechnology, #sc-271552) antibody overnight at 4 °C followed by exposure to goat anti-rabbit- IgG Alexa Fluor 488 or goat anti-mouse-IgG Alexa fluor 680 secondary antibody (Invitrogen; 1:500) for 1h at room temperature.

Techniques: Functional Assay, Immunoprecipitation, Control, Western Blot, Expressing, Plasmid Preparation