Structured Review

Novus Biologicals anti dog1 tmem16a
Expression of <t>TMEM16A</t> in human healthy, asthmatic, and CF lungs. Lung slices of normal airways indicate little expression of TMEM16A in both surface airway epithelium and airway submucosal glands. Pronounced expression of TMEM16A in apical membranes of surface epithelial cells and airway submucosal glands of patients with asthma and CF (brown precipitation, DAP staining). Submucosal glands were often found to be hypertrophic. Representative stainings of four patients each. Bars indicate 20 µm.
Anti Dog1 Tmem16a, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti dog1 tmem16a/product/Novus Biologicals
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti dog1 tmem16a - by Bioz Stars, 2023-01
93/100 stars

Images

1) Product Images from "Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease"

Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22157852

Expression of TMEM16A in human healthy, asthmatic, and CF lungs. Lung slices of normal airways indicate little expression of TMEM16A in both surface airway epithelium and airway submucosal glands. Pronounced expression of TMEM16A in apical membranes of surface epithelial cells and airway submucosal glands of patients with asthma and CF (brown precipitation, DAP staining). Submucosal glands were often found to be hypertrophic. Representative stainings of four patients each. Bars indicate 20 µm.
Figure Legend Snippet: Expression of TMEM16A in human healthy, asthmatic, and CF lungs. Lung slices of normal airways indicate little expression of TMEM16A in both surface airway epithelium and airway submucosal glands. Pronounced expression of TMEM16A in apical membranes of surface epithelial cells and airway submucosal glands of patients with asthma and CF (brown precipitation, DAP staining). Submucosal glands were often found to be hypertrophic. Representative stainings of four patients each. Bars indicate 20 µm.

Techniques Used: Expressing, Staining

Activation of TMEM16A by Eact induces mucus release and airway contraction. ( A ) Mucus staining by alcian blue in OVA-treated asthmatic mice. Acute application of the activator of TMEM16A, Eact (4.8 µg/mL intratracheal), induced acute release of mucus from goblet cells and increased intraluminal mucus. Bars indicate 50 µm. ( B ) Summary of intracellular and intraluminal mucus. ( C ) Summary of cross sectional area indicating airway contraction by Eact. Mean ± SEM (number of animals/number of measurements). # significant difference when compared to control ( p < 0.05, unpaired t -test).
Figure Legend Snippet: Activation of TMEM16A by Eact induces mucus release and airway contraction. ( A ) Mucus staining by alcian blue in OVA-treated asthmatic mice. Acute application of the activator of TMEM16A, Eact (4.8 µg/mL intratracheal), induced acute release of mucus from goblet cells and increased intraluminal mucus. Bars indicate 50 µm. ( B ) Summary of intracellular and intraluminal mucus. ( C ) Summary of cross sectional area indicating airway contraction by Eact. Mean ± SEM (number of animals/number of measurements). # significant difference when compared to control ( p < 0.05, unpaired t -test).

Techniques Used: Activation Assay, Staining

Expression of MUC5AC and activation of TMEM16A in Calu-3 airway epithelial cells is inhibited by niclosamide. ( A ) Expression of MUC5AC induced by IL-13 (20 ng/mL; 72 h) in Calu-3 airway epithelial cells was inhibited by simultaneous incubation with niclosamide (1 µM). Bar = 100 µm. ( B ) Quantification of MUC5AC expression indicating inhibition by niclosamide (Niclo). ( C ) Current overlays from whole-cell patch clamp experiments before and after induction of MUC5AC expression by IL-13. Activation of whole-cell currents by purinergic stimulation (ATP, 100 µM) was enhanced by IL-13, which was completely inhibited by acute application of niclosamide (1 µM). ( D ) Corresponding current/voltage relationships. The inhibitor of Ca 2+ -activated KCNN4 K + channels, TRAM-34 (100 nM), was present in all patch clamp experiments to avoid potential activation of Ca 2+ -activated K + channels. Mean ± SEM (number of cells). * significant activation by ATP ( p < 0.05, paired t -test). # significant difference when compared to the absence of IL-13 ( p < 0.05, unpaired t -test).
Figure Legend Snippet: Expression of MUC5AC and activation of TMEM16A in Calu-3 airway epithelial cells is inhibited by niclosamide. ( A ) Expression of MUC5AC induced by IL-13 (20 ng/mL; 72 h) in Calu-3 airway epithelial cells was inhibited by simultaneous incubation with niclosamide (1 µM). Bar = 100 µm. ( B ) Quantification of MUC5AC expression indicating inhibition by niclosamide (Niclo). ( C ) Current overlays from whole-cell patch clamp experiments before and after induction of MUC5AC expression by IL-13. Activation of whole-cell currents by purinergic stimulation (ATP, 100 µM) was enhanced by IL-13, which was completely inhibited by acute application of niclosamide (1 µM). ( D ) Corresponding current/voltage relationships. The inhibitor of Ca 2+ -activated KCNN4 K + channels, TRAM-34 (100 nM), was present in all patch clamp experiments to avoid potential activation of Ca 2+ -activated K + channels. Mean ± SEM (number of cells). * significant activation by ATP ( p < 0.05, paired t -test). # significant difference when compared to the absence of IL-13 ( p < 0.05, unpaired t -test).

Techniques Used: Expressing, Activation Assay, Incubation, Inhibition, Patch Clamp

Niclosamide inhibits expression of MUC5AC and SPDEF in Calu-3 cells. ( A ) Western blot indicating upregulation of TMEM16A in Calu-3 cells by IL-13 (20 ng/mL, 72 h) and inhibition of expression by niclosamide (1 µM). Blots were performed in triplicates. ( B ) RT-PCR analysis of the expression of MUC5AC, TMEM16A, and SAM pointed domain-containing ETS transcription factor (SPDEF) in Calu-3 airway epithelial cells. SPDEF is an integrator of goblet cell differentiation and pulmonary Th2 inflammation. ( C ) Low cycle numbers (20×) were chosen for quantification of expression by relating specific signals to expression of the housekeeper protein GAPDH. IL-13 (20 ng/mL) leads to upregulation of expression of MUC5AC, TMEM16A, and SPDEF. Niclosamide (Niclo, 1 µM, 72 h) strongly inhibits expression of MUC5AC, TMEM16A, and SPDEF. Mean ± SEM (number of assays). #,§ significant increase by IL-13 and inhibition by niclosamide, respectively ( p < 0.05, ANOVA).
Figure Legend Snippet: Niclosamide inhibits expression of MUC5AC and SPDEF in Calu-3 cells. ( A ) Western blot indicating upregulation of TMEM16A in Calu-3 cells by IL-13 (20 ng/mL, 72 h) and inhibition of expression by niclosamide (1 µM). Blots were performed in triplicates. ( B ) RT-PCR analysis of the expression of MUC5AC, TMEM16A, and SAM pointed domain-containing ETS transcription factor (SPDEF) in Calu-3 airway epithelial cells. SPDEF is an integrator of goblet cell differentiation and pulmonary Th2 inflammation. ( C ) Low cycle numbers (20×) were chosen for quantification of expression by relating specific signals to expression of the housekeeper protein GAPDH. IL-13 (20 ng/mL) leads to upregulation of expression of MUC5AC, TMEM16A, and SPDEF. Niclosamide (Niclo, 1 µM, 72 h) strongly inhibits expression of MUC5AC, TMEM16A, and SPDEF. Mean ± SEM (number of assays). #,§ significant increase by IL-13 and inhibition by niclosamide, respectively ( p < 0.05, ANOVA).

Techniques Used: Expressing, Western Blot, Inhibition, Reverse Transcription Polymerase Chain Reaction, Cell Differentiation

Activation of TMEM16A whole-cell currents is potentiated by brevenal. ( A ) Dose-dependent activation of endogenous TMEM16A whole-cell currents in CFBE airway epithelial cells, by the purinergic agonist ATP. Clamp voltages ± 100 mV in steps of 20 mV. ( B ) Corresponding current/voltage relationships. ( C , D ) Activation of whole-cell currents obtained from cells pre-incubated with brevenal (500 nM, 15 min) and corresponding current/voltage relationships. ( E ) Activation of whole-cell currents in brevenal-incubated cells, in which expression of TMEM16A has been knocked down by treatment with siRNA-TMEM16A (c.f. ). ( F ) Corresponding current/voltage relationships. Mean ± SEM (number of cells). * significant activation by ATP ( p < 0.05, ANOVA). The inhibitor of Ca 2+ -activated KCNN4 K + channels, TRAM-34 (100 nM), was present in all patch clamp experiments to avoid potential activation of Ca 2+ -activated K + channels. # significant difference when compared to the absence of brevenal ( p < 0.05, ANOVA). No currents were activated in siRNA-TMEM16A-treated cells.
Figure Legend Snippet: Activation of TMEM16A whole-cell currents is potentiated by brevenal. ( A ) Dose-dependent activation of endogenous TMEM16A whole-cell currents in CFBE airway epithelial cells, by the purinergic agonist ATP. Clamp voltages ± 100 mV in steps of 20 mV. ( B ) Corresponding current/voltage relationships. ( C , D ) Activation of whole-cell currents obtained from cells pre-incubated with brevenal (500 nM, 15 min) and corresponding current/voltage relationships. ( E ) Activation of whole-cell currents in brevenal-incubated cells, in which expression of TMEM16A has been knocked down by treatment with siRNA-TMEM16A (c.f. ). ( F ) Corresponding current/voltage relationships. Mean ± SEM (number of cells). * significant activation by ATP ( p < 0.05, ANOVA). The inhibitor of Ca 2+ -activated KCNN4 K + channels, TRAM-34 (100 nM), was present in all patch clamp experiments to avoid potential activation of Ca 2+ -activated K + channels. # significant difference when compared to the absence of brevenal ( p < 0.05, ANOVA). No currents were activated in siRNA-TMEM16A-treated cells.

Techniques Used: Activation Assay, Incubation, Expressing, Patch Clamp

RT-PCR Primer.
Figure Legend Snippet: RT-PCR Primer.

Techniques Used:


Structured Review

Novus Biologicals tmem16a
<t>Ano1</t> 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).
Tmem16a, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/tmem16a/product/Novus Biologicals
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
tmem16a - by Bioz Stars, 2023-01
93/100 stars

Images

1) Product Images from "Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner"

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

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2021.100912

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).
Figure Legend 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).

Techniques Used: Expressing, Incubation

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).
Figure Legend 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).

Techniques Used: Western Blot

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.)
Figure Legend 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.)

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

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.
Figure Legend 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.

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


Structured Review

Novus Biologicals rabbit monoclonal anti tmem16a dog1 antibody
<t>TMEM16A</t> is activated by Hypo and augments I Cl ,swell . (A) Continues recording of whole cell currents activated in HEK293 cells by hypotonic cell swelling (Hypo, 200 mosm/l). (B) Current/voltage relationships of Hypo-activated whole-cell currents. TMEM16A is activated by Hypo and further augments I Cl ,swell in cells coexpressing TMEM16A and LRRC8A/C. Mean ± SEM; n = 8–11 for each series). # Significant increase when compared to mock ( p < 0.05–0.0001; unpaired t -tests).
Rabbit Monoclonal Anti Tmem16a Dog1 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit monoclonal anti tmem16a dog1 antibody/product/Novus Biologicals
Average 92 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rabbit monoclonal anti tmem16a dog1 antibody - by Bioz Stars, 2023-01
92/100 stars

Images

1) Product Images from "Ca 2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl – Channels"

Article Title: Ca 2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl – Channels

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.596879

TMEM16A is activated by Hypo and augments I Cl ,swell . (A) Continues recording of whole cell currents activated in HEK293 cells by hypotonic cell swelling (Hypo, 200 mosm/l). (B) Current/voltage relationships of Hypo-activated whole-cell currents. TMEM16A is activated by Hypo and further augments I Cl ,swell in cells coexpressing TMEM16A and LRRC8A/C. Mean ± SEM; n = 8–11 for each series). # Significant increase when compared to mock ( p < 0.05–0.0001; unpaired t -tests).
Figure Legend Snippet: TMEM16A is activated by Hypo and augments I Cl ,swell . (A) Continues recording of whole cell currents activated in HEK293 cells by hypotonic cell swelling (Hypo, 200 mosm/l). (B) Current/voltage relationships of Hypo-activated whole-cell currents. TMEM16A is activated by Hypo and further augments I Cl ,swell in cells coexpressing TMEM16A and LRRC8A/C. Mean ± SEM; n = 8–11 for each series). # Significant increase when compared to mock ( p < 0.05–0.0001; unpaired t -tests).

Techniques Used:

TMEM16A overexpressed in HEK293 cells is spontaneously active. (A) Continuous current recordings of a mock-transfected and a TMEM16A-expressing HEK293 cell. In these experiments, the pipette filling solution and the bath solution contained CsCl (c.f. Methods). Cl – removal from the extracellular bath solution except of 5 mM (5Cl – ) had very little effect on the mock-transfected cell but strongly depolarized the TMEM16A-expressing cell. (B) Corresponding I/V curves indicate small currents and little effect of 5Cl – in mock-transfected cells, while currents were large and strongly inhibited with a shift of the reversal potential in TMEM16A-overexpressing cells.
Figure Legend Snippet: TMEM16A overexpressed in HEK293 cells is spontaneously active. (A) Continuous current recordings of a mock-transfected and a TMEM16A-expressing HEK293 cell. In these experiments, the pipette filling solution and the bath solution contained CsCl (c.f. Methods). Cl – removal from the extracellular bath solution except of 5 mM (5Cl – ) had very little effect on the mock-transfected cell but strongly depolarized the TMEM16A-expressing cell. (B) Corresponding I/V curves indicate small currents and little effect of 5Cl – in mock-transfected cells, while currents were large and strongly inhibited with a shift of the reversal potential in TMEM16A-overexpressing cells.

Techniques Used: Transfection, Expressing, Transferring

TMEM16A supports activation of LRRC8. (A) Hypo (200 mosm/l)-induced whole-cell current overlays obtained in HT 29 cells treated with scrambled RNA (scrbld), siRNA for TMEM16A, LRRC8A, or both. The free Ca 2+ concentration in the patch pipette filling solution was 100 nM. (B) Corresponding current–voltage relationships. (C) Hypo-induced whole-cell current densities (Vc = +100 mV). (D) Time-dependent activation of whole-cell currents by Hypo. Mean ± SEM; n = 9–14 for each series). # Significant decrease when compared to mock ( p < 0.01 for all; unpaired t -tests).
Figure Legend Snippet: TMEM16A supports activation of LRRC8. (A) Hypo (200 mosm/l)-induced whole-cell current overlays obtained in HT 29 cells treated with scrambled RNA (scrbld), siRNA for TMEM16A, LRRC8A, or both. The free Ca 2+ concentration in the patch pipette filling solution was 100 nM. (B) Corresponding current–voltage relationships. (C) Hypo-induced whole-cell current densities (Vc = +100 mV). (D) Time-dependent activation of whole-cell currents by Hypo. Mean ± SEM; n = 9–14 for each series). # Significant decrease when compared to mock ( p < 0.01 for all; unpaired t -tests).

Techniques Used: Activation Assay, Concentration Assay, Transferring

Hypo-induced anion permeability measured under non-voltage clamp conditions. (A) siRNA knockdown of LRRC8A detected by Western blotting. The knockdown efficiency was 72%. Knockdown of TMEM16A enhanced the expression of LRRC8A, corresponding to . (B) Summary of time dependence for YFP quenching induced by 20 mM iodide in the presence of isotonic Ringer (Iso; 300 mosmol/l) or Hypo (200 mosm/l). (C) Summary of initial rate of quenching (arbitrary units/s) ( n = 4; p < 0.006). (D) siRNA knockdown of TMEM16A detected by Western blotting. (E) Summary of time dependence for Hypo-induced YFP quenching in the absence or presence of siRNA. (F) Summary of initial rates of quenching (arbitrary units/s) ( n = 19). p < 0.0005 (siTMEM16A), p < 2 × 10 –11 (siLRRC8A), p < 2 × 10 –11 (siLRRC8A/siTMEM16A). (G,H) Hypo-induced YFP quenching was absent in the presence of 0 mM extracellular iodide but was gradually increased with 5 mM ( n = 4; p < 0.003), 20 mM ( n = 4; p < 0.0009), and 50 mM ( n = 4; p < 0.0005). Mean ± SEM; # significant increase or decrease, respectively (unpaired t -test). Blots were done as replicates.
Figure Legend Snippet: Hypo-induced anion permeability measured under non-voltage clamp conditions. (A) siRNA knockdown of LRRC8A detected by Western blotting. The knockdown efficiency was 72%. Knockdown of TMEM16A enhanced the expression of LRRC8A, corresponding to . (B) Summary of time dependence for YFP quenching induced by 20 mM iodide in the presence of isotonic Ringer (Iso; 300 mosmol/l) or Hypo (200 mosm/l). (C) Summary of initial rate of quenching (arbitrary units/s) ( n = 4; p < 0.006). (D) siRNA knockdown of TMEM16A detected by Western blotting. (E) Summary of time dependence for Hypo-induced YFP quenching in the absence or presence of siRNA. (F) Summary of initial rates of quenching (arbitrary units/s) ( n = 19). p < 0.0005 (siTMEM16A), p < 2 × 10 –11 (siLRRC8A), p < 2 × 10 –11 (siLRRC8A/siTMEM16A). (G,H) Hypo-induced YFP quenching was absent in the presence of 0 mM extracellular iodide but was gradually increased with 5 mM ( n = 4; p < 0.003), 20 mM ( n = 4; p < 0.0009), and 50 mM ( n = 4; p < 0.0005). Mean ± SEM; # significant increase or decrease, respectively (unpaired t -test). Blots were done as replicates.

Techniques Used: Permeability, Western Blot, Expressing

Hypotonicity activates TMEM16A-dependent Ca 2+ increase. (A) Summary of Hypo (200 mosm/l)-induced current densities inhibited by DCPIB in HT 29 cells ( n = 9–15). The free Ca 2+ concentration in the patch pipette filling solution was 100 nM. (B) Hypo-induced whole-cell current overlays in HT 29 cells treated with scrambled RNA (scrbld), siRNA for TMEM16A, or both TMEM16F. (C) Effect of 2-APB (50 μM; p < 0.001) and dantrolene (50 μM; p < 0.001) on Hypo-induced currents densities (Vc = +100 mV) ( n = 9–11). (D) ATP (100 μM)-induced whole-cell current overlays in the absence or presence of siRNAs. (E) Corresponding current–voltage relationships ( n = 9–11). (F) Effect of 2-APB (50 μM; p < 0.001) and dantrolene (50 μM) on ATP-induced current densities (Vc = +100 mV) ( n = 7–9). (G) Effect of TMEM16A-siRNA on ATP-induced Ca 2+ -increase ( n = 21–45; p < 0.000004 and p < 0.01, respectively). (H) Effect of TMEM16A-siRNA on Hypo-induced Ca 2+ -increase ( n = 21–55; p < 0.003). Plateau Ca 2+ was determined at the end of the plateau. Mean ± SEM; # significant decrease (unpaired t -test).
Figure Legend Snippet: Hypotonicity activates TMEM16A-dependent Ca 2+ increase. (A) Summary of Hypo (200 mosm/l)-induced current densities inhibited by DCPIB in HT 29 cells ( n = 9–15). The free Ca 2+ concentration in the patch pipette filling solution was 100 nM. (B) Hypo-induced whole-cell current overlays in HT 29 cells treated with scrambled RNA (scrbld), siRNA for TMEM16A, or both TMEM16F. (C) Effect of 2-APB (50 μM; p < 0.001) and dantrolene (50 μM; p < 0.001) on Hypo-induced currents densities (Vc = +100 mV) ( n = 9–11). (D) ATP (100 μM)-induced whole-cell current overlays in the absence or presence of siRNAs. (E) Corresponding current–voltage relationships ( n = 9–11). (F) Effect of 2-APB (50 μM; p < 0.001) and dantrolene (50 μM) on ATP-induced current densities (Vc = +100 mV) ( n = 7–9). (G) Effect of TMEM16A-siRNA on ATP-induced Ca 2+ -increase ( n = 21–45; p < 0.000004 and p < 0.01, respectively). (H) Effect of TMEM16A-siRNA on Hypo-induced Ca 2+ -increase ( n = 21–55; p < 0.003). Plateau Ca 2+ was determined at the end of the plateau. Mean ± SEM; # significant decrease (unpaired t -test).

Techniques Used: Concentration Assay, Transferring

Role of Ca 2+ signaling for Hypo- and ATP-induced anion permeability. (A) Time-dependent YFP quenching induced by different concentrations of ATP ( n = 3–5). (B) Concentration dependence of ATP-induced quenching (arbitrary units/s) and effects of knockdown of TMEM16A or LRRC8A ( n = 4). (C) Summary of ATP-induced quenching in the absence (control) or presence of IP3R blockers xestospongin C (10 μM; p < 10 –19 ), suramin (500 μM; p < 10 –7 ), and probenecid (1 mM; p < 2 × 10 –9 ) ( n = 4–5). (D) Summary of Hypo (200 mosm/l)-induced quenching in the absence (control) or presence of DCPIB (30 μM; p < 0.02), Ani9 (10 μM), suramin (500 μM; p < 2 × 10 –7 ), U73122 (10 μM; p < 0.01), 2-APB (50 μM; p < 10 –17 ), dantrolene (50 μM; p < 0.02), probenecid (1 mM; p < 0.03), and NS3728 (10 μM; p < 0.03) ( n = 4–5). (E,F) The contribution of Ca 2+ influx on Hypo-induced quenching was examined by exposing the cells to a hypotonic solution in the presence of physiological (1.3 mM; n = 5) or low (1 μM; n = 4; p < 0.02) extracellular Ca 2+ concentration. Mean ± SEM; # significant inhibition (unpaired t -test).
Figure Legend Snippet: Role of Ca 2+ signaling for Hypo- and ATP-induced anion permeability. (A) Time-dependent YFP quenching induced by different concentrations of ATP ( n = 3–5). (B) Concentration dependence of ATP-induced quenching (arbitrary units/s) and effects of knockdown of TMEM16A or LRRC8A ( n = 4). (C) Summary of ATP-induced quenching in the absence (control) or presence of IP3R blockers xestospongin C (10 μM; p < 10 –19 ), suramin (500 μM; p < 10 –7 ), and probenecid (1 mM; p < 2 × 10 –9 ) ( n = 4–5). (D) Summary of Hypo (200 mosm/l)-induced quenching in the absence (control) or presence of DCPIB (30 μM; p < 0.02), Ani9 (10 μM), suramin (500 μM; p < 2 × 10 –7 ), U73122 (10 μM; p < 0.01), 2-APB (50 μM; p < 10 –17 ), dantrolene (50 μM; p < 0.02), probenecid (1 mM; p < 0.03), and NS3728 (10 μM; p < 0.03) ( n = 4–5). (E,F) The contribution of Ca 2+ influx on Hypo-induced quenching was examined by exposing the cells to a hypotonic solution in the presence of physiological (1.3 mM; n = 5) or low (1 μM; n = 4; p < 0.02) extracellular Ca 2+ concentration. Mean ± SEM; # significant inhibition (unpaired t -test).

Techniques Used: Permeability, Concentration Assay, Inhibition


Structured Review

Novus Biologicals rabbit polyclonal anti tmem16a dog1 antibody
Endogenous Ca 2+ -dependent <t>TMEM16A</t> 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).
Rabbit Polyclonal Anti Tmem16a Dog1 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit polyclonal anti tmem16a dog1 antibody/product/Novus Biologicals
Average 92 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rabbit polyclonal anti tmem16a dog1 antibody - by Bioz Stars, 2023-01
92/100 stars

Images

1) Product Images from "Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A"

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

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21072557

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).
Figure Legend 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).

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

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).
Figure Legend 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).

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

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).
Figure Legend 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).

Techniques Used: Inhibition, Activation Assay

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).
Figure Legend 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).

Techniques Used: Transferring, Inhibition

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).
Figure Legend 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).

Techniques Used: Activation Assay

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

Techniques Used: Activation Assay

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).
Figure Legend 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).

Techniques Used: Transferring, Activation Assay

RT-PCR primers.
Figure Legend Snippet: RT-PCR primers.

Techniques Used:


Structured Review

Novus Biologicals rabbit polyclonal tmem16a antibody
Rabbit Polyclonal Tmem16a Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit polyclonal tmem16a antibody/product/Novus Biologicals
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
rabbit polyclonal tmem16a antibody - by Bioz Stars, 2023-01
93/100 stars

Images


Structured Review

Novus Biologicals anti tmem16a antibodies
Anti Tmem16a Antibodies, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti tmem16a antibodies/product/Novus Biologicals
Average 85 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti tmem16a antibodies - by Bioz Stars, 2023-01
85/100 stars

Images


Structured Review

Novus Biologicals anti tmem16a antibodies
Anti Tmem16a Antibodies, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti tmem16a antibodies/product/Novus Biologicals
Average 85 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti tmem16a antibodies - by Bioz Stars, 2023-01
85/100 stars

Images


Structured Review

Novus Biologicals tmem16a primary antibody
Tmem16a Primary Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/tmem16a primary antibody/product/Novus Biologicals
Average 85 stars, based on 1 article reviews
Price from $9.99 to $1999.99
tmem16a primary antibody - by Bioz Stars, 2023-01
85/100 stars

Images


Structured Review

Novus Biologicals nbp1 49559 mouse monoclonal
Nbp1 49559 Mouse Monoclonal, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/nbp1 49559 mouse monoclonal/product/Novus Biologicals
Average 85 stars, based on 1 article reviews
Price from $9.99 to $1999.99
nbp1 49559 mouse monoclonal - by Bioz Stars, 2023-01
85/100 stars

Images

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Novus Biologicals anti dog1 tmem16a
    Expression of <t>TMEM16A</t> in human healthy, asthmatic, and CF lungs. Lung slices of normal airways indicate little expression of TMEM16A in both surface airway epithelium and airway submucosal glands. Pronounced expression of TMEM16A in apical membranes of surface epithelial cells and airway submucosal glands of patients with asthma and CF (brown precipitation, DAP staining). Submucosal glands were often found to be hypertrophic. Representative stainings of four patients each. Bars indicate 20 µm.
    Anti Dog1 Tmem16a, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti dog1 tmem16a/product/Novus Biologicals
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti dog1 tmem16a - by Bioz Stars, 2023-01
    93/100 stars
      Buy from Supplier

    93
    Novus Biologicals tmem16a
    <t>Ano1</t> 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).
    Tmem16a, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tmem16a/product/Novus Biologicals
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tmem16a - by Bioz Stars, 2023-01
    93/100 stars
      Buy from Supplier

    92
    Novus Biologicals rabbit monoclonal anti tmem16a dog1 antibody
    <t>TMEM16A</t> is activated by Hypo and augments I Cl ,swell . (A) Continues recording of whole cell currents activated in HEK293 cells by hypotonic cell swelling (Hypo, 200 mosm/l). (B) Current/voltage relationships of Hypo-activated whole-cell currents. TMEM16A is activated by Hypo and further augments I Cl ,swell in cells coexpressing TMEM16A and LRRC8A/C. Mean ± SEM; n = 8–11 for each series). # Significant increase when compared to mock ( p < 0.05–0.0001; unpaired t -tests).
    Rabbit Monoclonal Anti Tmem16a Dog1 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal anti tmem16a dog1 antibody/product/Novus Biologicals
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit monoclonal anti tmem16a dog1 antibody - by Bioz Stars, 2023-01
    92/100 stars
      Buy from Supplier

    92
    Novus Biologicals rabbit polyclonal anti tmem16a dog1 antibody
    Endogenous Ca 2+ -dependent <t>TMEM16A</t> 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).
    Rabbit Polyclonal Anti Tmem16a Dog1 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti tmem16a dog1 antibody/product/Novus Biologicals
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti tmem16a dog1 antibody - by Bioz Stars, 2023-01
    92/100 stars
      Buy from Supplier

    93
    Novus Biologicals rabbit polyclonal tmem16a antibody
    Endogenous Ca 2+ -dependent <t>TMEM16A</t> 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).
    Rabbit Polyclonal Tmem16a Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal tmem16a antibody/product/Novus Biologicals
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal tmem16a antibody - by Bioz Stars, 2023-01
    93/100 stars
      Buy from Supplier

    85
    Novus Biologicals anti tmem16a antibodies
    Endogenous Ca 2+ -dependent <t>TMEM16A</t> 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).
    Anti Tmem16a Antibodies, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti tmem16a antibodies/product/Novus Biologicals
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti tmem16a antibodies - by Bioz Stars, 2023-01
    85/100 stars
      Buy from Supplier

    85
    Novus Biologicals tmem16a primary antibody
    Endogenous Ca 2+ -dependent <t>TMEM16A</t> 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).
    Tmem16a Primary Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tmem16a primary antibody/product/Novus Biologicals
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tmem16a primary antibody - by Bioz Stars, 2023-01
    85/100 stars
      Buy from Supplier

    85
    Novus Biologicals nbp1 49559 mouse monoclonal
    Endogenous Ca 2+ -dependent <t>TMEM16A</t> 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).
    Nbp1 49559 Mouse Monoclonal, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nbp1 49559 mouse monoclonal/product/Novus Biologicals
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nbp1 49559 mouse monoclonal - by Bioz Stars, 2023-01
    85/100 stars
      Buy from Supplier

    Image Search Results


    Expression of TMEM16A in human healthy, asthmatic, and CF lungs. Lung slices of normal airways indicate little expression of TMEM16A in both surface airway epithelium and airway submucosal glands. Pronounced expression of TMEM16A in apical membranes of surface epithelial cells and airway submucosal glands of patients with asthma and CF (brown precipitation, DAP staining). Submucosal glands were often found to be hypertrophic. Representative stainings of four patients each. Bars indicate 20 µm.

    Journal: International Journal of Molecular Sciences

    Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

    doi: 10.3390/ijms22157852

    Figure Lengend Snippet: Expression of TMEM16A in human healthy, asthmatic, and CF lungs. Lung slices of normal airways indicate little expression of TMEM16A in both surface airway epithelium and airway submucosal glands. Pronounced expression of TMEM16A in apical membranes of surface epithelial cells and airway submucosal glands of patients with asthma and CF (brown precipitation, DAP staining). Submucosal glands were often found to be hypertrophic. Representative stainings of four patients each. Bars indicate 20 µm.

    Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

    Techniques: Expressing, Staining

    Activation of TMEM16A by Eact induces mucus release and airway contraction. ( A ) Mucus staining by alcian blue in OVA-treated asthmatic mice. Acute application of the activator of TMEM16A, Eact (4.8 µg/mL intratracheal), induced acute release of mucus from goblet cells and increased intraluminal mucus. Bars indicate 50 µm. ( B ) Summary of intracellular and intraluminal mucus. ( C ) Summary of cross sectional area indicating airway contraction by Eact. Mean ± SEM (number of animals/number of measurements). # significant difference when compared to control ( p < 0.05, unpaired t -test).

    Journal: International Journal of Molecular Sciences

    Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

    doi: 10.3390/ijms22157852

    Figure Lengend Snippet: Activation of TMEM16A by Eact induces mucus release and airway contraction. ( A ) Mucus staining by alcian blue in OVA-treated asthmatic mice. Acute application of the activator of TMEM16A, Eact (4.8 µg/mL intratracheal), induced acute release of mucus from goblet cells and increased intraluminal mucus. Bars indicate 50 µm. ( B ) Summary of intracellular and intraluminal mucus. ( C ) Summary of cross sectional area indicating airway contraction by Eact. Mean ± SEM (number of animals/number of measurements). # significant difference when compared to control ( p < 0.05, unpaired t -test).

    Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

    Techniques: Activation Assay, Staining

    Expression of MUC5AC and activation of TMEM16A in Calu-3 airway epithelial cells is inhibited by niclosamide. ( A ) Expression of MUC5AC induced by IL-13 (20 ng/mL; 72 h) in Calu-3 airway epithelial cells was inhibited by simultaneous incubation with niclosamide (1 µM). Bar = 100 µm. ( B ) Quantification of MUC5AC expression indicating inhibition by niclosamide (Niclo). ( C ) Current overlays from whole-cell patch clamp experiments before and after induction of MUC5AC expression by IL-13. Activation of whole-cell currents by purinergic stimulation (ATP, 100 µM) was enhanced by IL-13, which was completely inhibited by acute application of niclosamide (1 µM). ( D ) Corresponding current/voltage relationships. The inhibitor of Ca 2+ -activated KCNN4 K + channels, TRAM-34 (100 nM), was present in all patch clamp experiments to avoid potential activation of Ca 2+ -activated K + channels. Mean ± SEM (number of cells). * significant activation by ATP ( p < 0.05, paired t -test). # significant difference when compared to the absence of IL-13 ( p < 0.05, unpaired t -test).

    Journal: International Journal of Molecular Sciences

    Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

    doi: 10.3390/ijms22157852

    Figure Lengend Snippet: Expression of MUC5AC and activation of TMEM16A in Calu-3 airway epithelial cells is inhibited by niclosamide. ( A ) Expression of MUC5AC induced by IL-13 (20 ng/mL; 72 h) in Calu-3 airway epithelial cells was inhibited by simultaneous incubation with niclosamide (1 µM). Bar = 100 µm. ( B ) Quantification of MUC5AC expression indicating inhibition by niclosamide (Niclo). ( C ) Current overlays from whole-cell patch clamp experiments before and after induction of MUC5AC expression by IL-13. Activation of whole-cell currents by purinergic stimulation (ATP, 100 µM) was enhanced by IL-13, which was completely inhibited by acute application of niclosamide (1 µM). ( D ) Corresponding current/voltage relationships. The inhibitor of Ca 2+ -activated KCNN4 K + channels, TRAM-34 (100 nM), was present in all patch clamp experiments to avoid potential activation of Ca 2+ -activated K + channels. Mean ± SEM (number of cells). * significant activation by ATP ( p < 0.05, paired t -test). # significant difference when compared to the absence of IL-13 ( p < 0.05, unpaired t -test).

    Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

    Techniques: Expressing, Activation Assay, Incubation, Inhibition, Patch Clamp

    Niclosamide inhibits expression of MUC5AC and SPDEF in Calu-3 cells. ( A ) Western blot indicating upregulation of TMEM16A in Calu-3 cells by IL-13 (20 ng/mL, 72 h) and inhibition of expression by niclosamide (1 µM). Blots were performed in triplicates. ( B ) RT-PCR analysis of the expression of MUC5AC, TMEM16A, and SAM pointed domain-containing ETS transcription factor (SPDEF) in Calu-3 airway epithelial cells. SPDEF is an integrator of goblet cell differentiation and pulmonary Th2 inflammation. ( C ) Low cycle numbers (20×) were chosen for quantification of expression by relating specific signals to expression of the housekeeper protein GAPDH. IL-13 (20 ng/mL) leads to upregulation of expression of MUC5AC, TMEM16A, and SPDEF. Niclosamide (Niclo, 1 µM, 72 h) strongly inhibits expression of MUC5AC, TMEM16A, and SPDEF. Mean ± SEM (number of assays). #,§ significant increase by IL-13 and inhibition by niclosamide, respectively ( p < 0.05, ANOVA).

    Journal: International Journal of Molecular Sciences

    Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

    doi: 10.3390/ijms22157852

    Figure Lengend Snippet: Niclosamide inhibits expression of MUC5AC and SPDEF in Calu-3 cells. ( A ) Western blot indicating upregulation of TMEM16A in Calu-3 cells by IL-13 (20 ng/mL, 72 h) and inhibition of expression by niclosamide (1 µM). Blots were performed in triplicates. ( B ) RT-PCR analysis of the expression of MUC5AC, TMEM16A, and SAM pointed domain-containing ETS transcription factor (SPDEF) in Calu-3 airway epithelial cells. SPDEF is an integrator of goblet cell differentiation and pulmonary Th2 inflammation. ( C ) Low cycle numbers (20×) were chosen for quantification of expression by relating specific signals to expression of the housekeeper protein GAPDH. IL-13 (20 ng/mL) leads to upregulation of expression of MUC5AC, TMEM16A, and SPDEF. Niclosamide (Niclo, 1 µM, 72 h) strongly inhibits expression of MUC5AC, TMEM16A, and SPDEF. Mean ± SEM (number of assays). #,§ significant increase by IL-13 and inhibition by niclosamide, respectively ( p < 0.05, ANOVA).

    Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

    Techniques: Expressing, Western Blot, Inhibition, Reverse Transcription Polymerase Chain Reaction, Cell Differentiation

    Activation of TMEM16A whole-cell currents is potentiated by brevenal. ( A ) Dose-dependent activation of endogenous TMEM16A whole-cell currents in CFBE airway epithelial cells, by the purinergic agonist ATP. Clamp voltages ± 100 mV in steps of 20 mV. ( B ) Corresponding current/voltage relationships. ( C , D ) Activation of whole-cell currents obtained from cells pre-incubated with brevenal (500 nM, 15 min) and corresponding current/voltage relationships. ( E ) Activation of whole-cell currents in brevenal-incubated cells, in which expression of TMEM16A has been knocked down by treatment with siRNA-TMEM16A (c.f. ). ( F ) Corresponding current/voltage relationships. Mean ± SEM (number of cells). * significant activation by ATP ( p < 0.05, ANOVA). The inhibitor of Ca 2+ -activated KCNN4 K + channels, TRAM-34 (100 nM), was present in all patch clamp experiments to avoid potential activation of Ca 2+ -activated K + channels. # significant difference when compared to the absence of brevenal ( p < 0.05, ANOVA). No currents were activated in siRNA-TMEM16A-treated cells.

    Journal: International Journal of Molecular Sciences

    Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

    doi: 10.3390/ijms22157852

    Figure Lengend Snippet: Activation of TMEM16A whole-cell currents is potentiated by brevenal. ( A ) Dose-dependent activation of endogenous TMEM16A whole-cell currents in CFBE airway epithelial cells, by the purinergic agonist ATP. Clamp voltages ± 100 mV in steps of 20 mV. ( B ) Corresponding current/voltage relationships. ( C , D ) Activation of whole-cell currents obtained from cells pre-incubated with brevenal (500 nM, 15 min) and corresponding current/voltage relationships. ( E ) Activation of whole-cell currents in brevenal-incubated cells, in which expression of TMEM16A has been knocked down by treatment with siRNA-TMEM16A (c.f. ). ( F ) Corresponding current/voltage relationships. Mean ± SEM (number of cells). * significant activation by ATP ( p < 0.05, ANOVA). The inhibitor of Ca 2+ -activated KCNN4 K + channels, TRAM-34 (100 nM), was present in all patch clamp experiments to avoid potential activation of Ca 2+ -activated K + channels. # significant difference when compared to the absence of brevenal ( p < 0.05, ANOVA). No currents were activated in siRNA-TMEM16A-treated cells.

    Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

    Techniques: Activation Assay, Incubation, Expressing, Patch Clamp

    RT-PCR Primer.

    Journal: International Journal of Molecular Sciences

    Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

    doi: 10.3390/ijms22157852

    Figure Lengend Snippet: RT-PCR Primer.

    Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

    Techniques:

    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).

    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: Expressing, Incubation

    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).

    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

    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.)

    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: Transfection, Western Blot, Expressing, Immunofluorescence, Co-Immunoprecipitation Assay

    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.

    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, Western Blot, Expressing, Plasmid Preparation

    TMEM16A is activated by Hypo and augments I Cl ,swell . (A) Continues recording of whole cell currents activated in HEK293 cells by hypotonic cell swelling (Hypo, 200 mosm/l). (B) Current/voltage relationships of Hypo-activated whole-cell currents. TMEM16A is activated by Hypo and further augments I Cl ,swell in cells coexpressing TMEM16A and LRRC8A/C. Mean ± SEM; n = 8–11 for each series). # Significant increase when compared to mock ( p < 0.05–0.0001; unpaired t -tests).

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Ca 2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl – Channels

    doi: 10.3389/fcell.2020.596879

    Figure Lengend Snippet: TMEM16A is activated by Hypo and augments I Cl ,swell . (A) Continues recording of whole cell currents activated in HEK293 cells by hypotonic cell swelling (Hypo, 200 mosm/l). (B) Current/voltage relationships of Hypo-activated whole-cell currents. TMEM16A is activated by Hypo and further augments I Cl ,swell in cells coexpressing TMEM16A and LRRC8A/C. Mean ± SEM; n = 8–11 for each series). # Significant increase when compared to mock ( p < 0.05–0.0001; unpaired t -tests).

    Article Snippet: Membranes were incubated with primary rabbit monoclonal anti-TMEM16A/DOG1 antibody (#NBP1-49799; Novus Biologicals, Littleton, Colorado, United States; 1:500 in 1% (w/v) NFM/TBS-T) or rabbit polyclonal anti-LRRC8A antibody (#HPA016811; Sigma-Aldrich, St. Louis, Missouri, United States; 1:1,000 in 1% (w/v) BSA/TBS-T) for 2.5 h at room temperature.

    Techniques:

    TMEM16A overexpressed in HEK293 cells is spontaneously active. (A) Continuous current recordings of a mock-transfected and a TMEM16A-expressing HEK293 cell. In these experiments, the pipette filling solution and the bath solution contained CsCl (c.f. Methods). Cl – removal from the extracellular bath solution except of 5 mM (5Cl – ) had very little effect on the mock-transfected cell but strongly depolarized the TMEM16A-expressing cell. (B) Corresponding I/V curves indicate small currents and little effect of 5Cl – in mock-transfected cells, while currents were large and strongly inhibited with a shift of the reversal potential in TMEM16A-overexpressing cells.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Ca 2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl – Channels

    doi: 10.3389/fcell.2020.596879

    Figure Lengend Snippet: TMEM16A overexpressed in HEK293 cells is spontaneously active. (A) Continuous current recordings of a mock-transfected and a TMEM16A-expressing HEK293 cell. In these experiments, the pipette filling solution and the bath solution contained CsCl (c.f. Methods). Cl – removal from the extracellular bath solution except of 5 mM (5Cl – ) had very little effect on the mock-transfected cell but strongly depolarized the TMEM16A-expressing cell. (B) Corresponding I/V curves indicate small currents and little effect of 5Cl – in mock-transfected cells, while currents were large and strongly inhibited with a shift of the reversal potential in TMEM16A-overexpressing cells.

    Article Snippet: Membranes were incubated with primary rabbit monoclonal anti-TMEM16A/DOG1 antibody (#NBP1-49799; Novus Biologicals, Littleton, Colorado, United States; 1:500 in 1% (w/v) NFM/TBS-T) or rabbit polyclonal anti-LRRC8A antibody (#HPA016811; Sigma-Aldrich, St. Louis, Missouri, United States; 1:1,000 in 1% (w/v) BSA/TBS-T) for 2.5 h at room temperature.

    Techniques: Transfection, Expressing, Transferring

    TMEM16A supports activation of LRRC8. (A) Hypo (200 mosm/l)-induced whole-cell current overlays obtained in HT 29 cells treated with scrambled RNA (scrbld), siRNA for TMEM16A, LRRC8A, or both. The free Ca 2+ concentration in the patch pipette filling solution was 100 nM. (B) Corresponding current–voltage relationships. (C) Hypo-induced whole-cell current densities (Vc = +100 mV). (D) Time-dependent activation of whole-cell currents by Hypo. Mean ± SEM; n = 9–14 for each series). # Significant decrease when compared to mock ( p < 0.01 for all; unpaired t -tests).

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Ca 2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl – Channels

    doi: 10.3389/fcell.2020.596879

    Figure Lengend Snippet: TMEM16A supports activation of LRRC8. (A) Hypo (200 mosm/l)-induced whole-cell current overlays obtained in HT 29 cells treated with scrambled RNA (scrbld), siRNA for TMEM16A, LRRC8A, or both. The free Ca 2+ concentration in the patch pipette filling solution was 100 nM. (B) Corresponding current–voltage relationships. (C) Hypo-induced whole-cell current densities (Vc = +100 mV). (D) Time-dependent activation of whole-cell currents by Hypo. Mean ± SEM; n = 9–14 for each series). # Significant decrease when compared to mock ( p < 0.01 for all; unpaired t -tests).

    Article Snippet: Membranes were incubated with primary rabbit monoclonal anti-TMEM16A/DOG1 antibody (#NBP1-49799; Novus Biologicals, Littleton, Colorado, United States; 1:500 in 1% (w/v) NFM/TBS-T) or rabbit polyclonal anti-LRRC8A antibody (#HPA016811; Sigma-Aldrich, St. Louis, Missouri, United States; 1:1,000 in 1% (w/v) BSA/TBS-T) for 2.5 h at room temperature.

    Techniques: Activation Assay, Concentration Assay, Transferring

    Hypo-induced anion permeability measured under non-voltage clamp conditions. (A) siRNA knockdown of LRRC8A detected by Western blotting. The knockdown efficiency was 72%. Knockdown of TMEM16A enhanced the expression of LRRC8A, corresponding to . (B) Summary of time dependence for YFP quenching induced by 20 mM iodide in the presence of isotonic Ringer (Iso; 300 mosmol/l) or Hypo (200 mosm/l). (C) Summary of initial rate of quenching (arbitrary units/s) ( n = 4; p < 0.006). (D) siRNA knockdown of TMEM16A detected by Western blotting. (E) Summary of time dependence for Hypo-induced YFP quenching in the absence or presence of siRNA. (F) Summary of initial rates of quenching (arbitrary units/s) ( n = 19). p < 0.0005 (siTMEM16A), p < 2 × 10 –11 (siLRRC8A), p < 2 × 10 –11 (siLRRC8A/siTMEM16A). (G,H) Hypo-induced YFP quenching was absent in the presence of 0 mM extracellular iodide but was gradually increased with 5 mM ( n = 4; p < 0.003), 20 mM ( n = 4; p < 0.0009), and 50 mM ( n = 4; p < 0.0005). Mean ± SEM; # significant increase or decrease, respectively (unpaired t -test). Blots were done as replicates.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Ca 2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl – Channels

    doi: 10.3389/fcell.2020.596879

    Figure Lengend Snippet: Hypo-induced anion permeability measured under non-voltage clamp conditions. (A) siRNA knockdown of LRRC8A detected by Western blotting. The knockdown efficiency was 72%. Knockdown of TMEM16A enhanced the expression of LRRC8A, corresponding to . (B) Summary of time dependence for YFP quenching induced by 20 mM iodide in the presence of isotonic Ringer (Iso; 300 mosmol/l) or Hypo (200 mosm/l). (C) Summary of initial rate of quenching (arbitrary units/s) ( n = 4; p < 0.006). (D) siRNA knockdown of TMEM16A detected by Western blotting. (E) Summary of time dependence for Hypo-induced YFP quenching in the absence or presence of siRNA. (F) Summary of initial rates of quenching (arbitrary units/s) ( n = 19). p < 0.0005 (siTMEM16A), p < 2 × 10 –11 (siLRRC8A), p < 2 × 10 –11 (siLRRC8A/siTMEM16A). (G,H) Hypo-induced YFP quenching was absent in the presence of 0 mM extracellular iodide but was gradually increased with 5 mM ( n = 4; p < 0.003), 20 mM ( n = 4; p < 0.0009), and 50 mM ( n = 4; p < 0.0005). Mean ± SEM; # significant increase or decrease, respectively (unpaired t -test). Blots were done as replicates.

    Article Snippet: Membranes were incubated with primary rabbit monoclonal anti-TMEM16A/DOG1 antibody (#NBP1-49799; Novus Biologicals, Littleton, Colorado, United States; 1:500 in 1% (w/v) NFM/TBS-T) or rabbit polyclonal anti-LRRC8A antibody (#HPA016811; Sigma-Aldrich, St. Louis, Missouri, United States; 1:1,000 in 1% (w/v) BSA/TBS-T) for 2.5 h at room temperature.

    Techniques: Permeability, Western Blot, Expressing

    Hypotonicity activates TMEM16A-dependent Ca 2+ increase. (A) Summary of Hypo (200 mosm/l)-induced current densities inhibited by DCPIB in HT 29 cells ( n = 9–15). The free Ca 2+ concentration in the patch pipette filling solution was 100 nM. (B) Hypo-induced whole-cell current overlays in HT 29 cells treated with scrambled RNA (scrbld), siRNA for TMEM16A, or both TMEM16F. (C) Effect of 2-APB (50 μM; p < 0.001) and dantrolene (50 μM; p < 0.001) on Hypo-induced currents densities (Vc = +100 mV) ( n = 9–11). (D) ATP (100 μM)-induced whole-cell current overlays in the absence or presence of siRNAs. (E) Corresponding current–voltage relationships ( n = 9–11). (F) Effect of 2-APB (50 μM; p < 0.001) and dantrolene (50 μM) on ATP-induced current densities (Vc = +100 mV) ( n = 7–9). (G) Effect of TMEM16A-siRNA on ATP-induced Ca 2+ -increase ( n = 21–45; p < 0.000004 and p < 0.01, respectively). (H) Effect of TMEM16A-siRNA on Hypo-induced Ca 2+ -increase ( n = 21–55; p < 0.003). Plateau Ca 2+ was determined at the end of the plateau. Mean ± SEM; # significant decrease (unpaired t -test).

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Ca 2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl – Channels

    doi: 10.3389/fcell.2020.596879

    Figure Lengend Snippet: Hypotonicity activates TMEM16A-dependent Ca 2+ increase. (A) Summary of Hypo (200 mosm/l)-induced current densities inhibited by DCPIB in HT 29 cells ( n = 9–15). The free Ca 2+ concentration in the patch pipette filling solution was 100 nM. (B) Hypo-induced whole-cell current overlays in HT 29 cells treated with scrambled RNA (scrbld), siRNA for TMEM16A, or both TMEM16F. (C) Effect of 2-APB (50 μM; p < 0.001) and dantrolene (50 μM; p < 0.001) on Hypo-induced currents densities (Vc = +100 mV) ( n = 9–11). (D) ATP (100 μM)-induced whole-cell current overlays in the absence or presence of siRNAs. (E) Corresponding current–voltage relationships ( n = 9–11). (F) Effect of 2-APB (50 μM; p < 0.001) and dantrolene (50 μM) on ATP-induced current densities (Vc = +100 mV) ( n = 7–9). (G) Effect of TMEM16A-siRNA on ATP-induced Ca 2+ -increase ( n = 21–45; p < 0.000004 and p < 0.01, respectively). (H) Effect of TMEM16A-siRNA on Hypo-induced Ca 2+ -increase ( n = 21–55; p < 0.003). Plateau Ca 2+ was determined at the end of the plateau. Mean ± SEM; # significant decrease (unpaired t -test).

    Article Snippet: Membranes were incubated with primary rabbit monoclonal anti-TMEM16A/DOG1 antibody (#NBP1-49799; Novus Biologicals, Littleton, Colorado, United States; 1:500 in 1% (w/v) NFM/TBS-T) or rabbit polyclonal anti-LRRC8A antibody (#HPA016811; Sigma-Aldrich, St. Louis, Missouri, United States; 1:1,000 in 1% (w/v) BSA/TBS-T) for 2.5 h at room temperature.

    Techniques: Concentration Assay, Transferring

    Role of Ca 2+ signaling for Hypo- and ATP-induced anion permeability. (A) Time-dependent YFP quenching induced by different concentrations of ATP ( n = 3–5). (B) Concentration dependence of ATP-induced quenching (arbitrary units/s) and effects of knockdown of TMEM16A or LRRC8A ( n = 4). (C) Summary of ATP-induced quenching in the absence (control) or presence of IP3R blockers xestospongin C (10 μM; p < 10 –19 ), suramin (500 μM; p < 10 –7 ), and probenecid (1 mM; p < 2 × 10 –9 ) ( n = 4–5). (D) Summary of Hypo (200 mosm/l)-induced quenching in the absence (control) or presence of DCPIB (30 μM; p < 0.02), Ani9 (10 μM), suramin (500 μM; p < 2 × 10 –7 ), U73122 (10 μM; p < 0.01), 2-APB (50 μM; p < 10 –17 ), dantrolene (50 μM; p < 0.02), probenecid (1 mM; p < 0.03), and NS3728 (10 μM; p < 0.03) ( n = 4–5). (E,F) The contribution of Ca 2+ influx on Hypo-induced quenching was examined by exposing the cells to a hypotonic solution in the presence of physiological (1.3 mM; n = 5) or low (1 μM; n = 4; p < 0.02) extracellular Ca 2+ concentration. Mean ± SEM; # significant inhibition (unpaired t -test).

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Ca 2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl – Channels

    doi: 10.3389/fcell.2020.596879

    Figure Lengend Snippet: Role of Ca 2+ signaling for Hypo- and ATP-induced anion permeability. (A) Time-dependent YFP quenching induced by different concentrations of ATP ( n = 3–5). (B) Concentration dependence of ATP-induced quenching (arbitrary units/s) and effects of knockdown of TMEM16A or LRRC8A ( n = 4). (C) Summary of ATP-induced quenching in the absence (control) or presence of IP3R blockers xestospongin C (10 μM; p < 10 –19 ), suramin (500 μM; p < 10 –7 ), and probenecid (1 mM; p < 2 × 10 –9 ) ( n = 4–5). (D) Summary of Hypo (200 mosm/l)-induced quenching in the absence (control) or presence of DCPIB (30 μM; p < 0.02), Ani9 (10 μM), suramin (500 μM; p < 2 × 10 –7 ), U73122 (10 μM; p < 0.01), 2-APB (50 μM; p < 10 –17 ), dantrolene (50 μM; p < 0.02), probenecid (1 mM; p < 0.03), and NS3728 (10 μM; p < 0.03) ( n = 4–5). (E,F) The contribution of Ca 2+ influx on Hypo-induced quenching was examined by exposing the cells to a hypotonic solution in the presence of physiological (1.3 mM; n = 5) or low (1 μM; n = 4; p < 0.02) extracellular Ca 2+ concentration. Mean ± SEM; # significant inhibition (unpaired t -test).

    Article Snippet: Membranes were incubated with primary rabbit monoclonal anti-TMEM16A/DOG1 antibody (#NBP1-49799; Novus Biologicals, Littleton, Colorado, United States; 1:500 in 1% (w/v) NFM/TBS-T) or rabbit polyclonal anti-LRRC8A antibody (#HPA016811; Sigma-Aldrich, St. Louis, Missouri, United States; 1:1,000 in 1% (w/v) BSA/TBS-T) for 2.5 h at room temperature.

    Techniques: Permeability, Concentration Assay, Inhibition

    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).

    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, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Activation Assay, Patch Clamp, Inhibition

    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).

    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, Inhibition

    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).

    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

    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).

    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, Inhibition

    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).

    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

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

    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

    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).

    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

    RT-PCR primers.

    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: