Journal: International Journal of Molecular Sciences

Article Title: Gender-Dependent Phenotype in Polycystic Kidney Disease Is Determined by Differential Intracellular Ca 2+ Signals

doi: 10.3390/ijms22116019

Figure Lengend Snippet: Expression of TMEM16A in male and female PKD1 −/− mice. ( A , B ) Western blotting of TMEM16A-expression in primary renal epithelial cells from kidneys of male and female Pkd1 +/+ and Pkd1 −/− mice. ( C ) Immunocytochemistry of Tmem16a in kidneys from male and female Pkd1 −/− mice. Bar = 20 µm. Representative images from three mice each. Mean ± SEM (number of animals in each series). # significant difference when compared to Pkd1 +/+ ( p < 0.05; unpaired t-test).

Article Snippet: Membranes were incubated with primary anti-TMEM16A (rabbit 1:500; Alomone, Jerusalem, Israel) mouse antibody overnight at 4 °C.

Techniques: Expressing, Western Blot, Immunocytochemistry

Journal: International Journal of Molecular Sciences

Article Title: Gender-Dependent Phenotype in Polycystic Kidney Disease Is Determined by Differential Intracellular Ca 2+ Signals

doi: 10.3390/ijms22116019

Figure Lengend Snippet: Effect of dihydrotestosterone on whole cell currents and intracellular Ca 2+ concentrations in mouse mCCDcl1 cortical collecting duct cells. ( A , B ) RT-PCR analysis of expression of Tmem16a, Cftr, androgen receptors ( A ), and estrogen receptors ( B ) in Pkd1 +/+ mCCDcl1 mouse collecting duct cells. ( C ) Western blot from mCCDcl1 cells, indicating upregulation of Tmem16a-expression by DHT. ( D , E ) Whole cell current overlays ( D ), corresponding I/V-curves ( E ), and summary of ATP (0.1 µM) -activated whole cell currents ( F ) from mCCDcl1 cells, indicating larger basal and ATP-activated currents in DHT (10 µM; 24 h) treated cells. Current densities (pA/pF) were assessed at the clamp voltage of +100 mV. ( G ) Basal and ATP (0.1–100 µM)–induced increase in intracellular Ca 2+ concentration in control (con) and DHT-incubated mCCDcl1 cells. Mean ± SEM (number of experiments). * Significant activation by ATP ( p < 0.05; paired t-test). # Significant difference compared to con ( p < 0.05; unpaired t-test).

Article Snippet: Membranes were incubated with primary anti-TMEM16A (rabbit 1:500; Alomone, Jerusalem, Israel) mouse antibody overnight at 4 °C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Concentration Assay, Incubation, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Gender-Dependent Phenotype in Polycystic Kidney Disease Is Determined by Differential Intracellular Ca 2+ Signals

doi: 10.3390/ijms22116019

Figure Lengend Snippet: Primers used for PCR-analysis.

Article Snippet: Membranes were incubated with primary anti-TMEM16A (rabbit 1:500; Alomone, Jerusalem, Israel) mouse antibody overnight at 4 °C.

Techniques:

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Blocking Assay, Knockdown, Control

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Produced, Concentration Assay

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Functional Assay, Control

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Blocking Assay, Control

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Imaging, Labeling

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Membrane, Activation Assay

Journal: Molecular medicine reports

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

doi: 10.3892/mmr.2016.4979

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

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

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

Journal: Molecular medicine reports

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

doi: 10.3892/mmr.2016.4979

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

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

Techniques: Patch Clamp, Concentration Assay

Journal: Molecular medicine reports

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

doi: 10.3892/mmr.2016.4979

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

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

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

Journal: Molecular medicine reports

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

doi: 10.3892/mmr.2016.4979

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

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

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

Journal: Molecular medicine reports

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

doi: 10.3892/mmr.2016.4979

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

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

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

Journal: Biochemistry and Biophysics Reports

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

doi: 10.1016/j.bbrep.2021.100912

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

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

Techniques: Gene Expression, Incubation

Journal: Biochemistry and Biophysics Reports

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

doi: 10.1016/j.bbrep.2021.100912

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

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

Techniques: Western Blot

Journal: Biochemistry and Biophysics Reports

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

doi: 10.1016/j.bbrep.2021.100912

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

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

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

Journal: Biochemistry and Biophysics Reports

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

doi: 10.1016/j.bbrep.2021.100912

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

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

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

Journal: PeerJ

Article Title: DOG1 is commonly expressed in pancreatic adenocarcinoma but unrelated to cancer aggressiveness

doi: 10.7717/peerj.11905

Figure Lengend Snippet: (A–D) DOG1 positive ductal adenocarcinomas with strong (A), moderate (B), focal moderate (C), and weak immunostaining of tumor cells (D). (A) and (C) also contain DOG1 negative normal ducts (arrow). (E & F) DOG1 negative cancers with diffuse (E) and periductal DOG1 (F) staining of stromal cells (F).

Article Snippet: Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking Solution (#52023; Agilent, Santa Clara, CA, USA) for 10 min. Anti-DOG1 mouse monoclonal antibody MSVA-201M (MS Validated Antibodies, Hamburg, Germany) was applied at 37 °C, 60 min, at 1:150.

Techniques: Immunostaining, Staining

Journal: PeerJ

Article Title: DOG1 is commonly expressed in pancreatic adenocarcinoma but unrelated to cancer aggressiveness

doi: 10.7717/peerj.11905

Figure Lengend Snippet: DOG1 immunostaining in cancer cells and cancer phenotype.

Article Snippet: Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking Solution (#52023; Agilent, Santa Clara, CA, USA) for 10 min. Anti-DOG1 mouse monoclonal antibody MSVA-201M (MS Validated Antibodies, Hamburg, Germany) was applied at 37 °C, 60 min, at 1:150.

Techniques: Immunostaining

Journal: PeerJ

Article Title: DOG1 is commonly expressed in pancreatic adenocarcinoma but unrelated to cancer aggressiveness

doi: 10.7717/peerj.11905

Figure Lengend Snippet: DOG1 immunostaining in stroma cells and cancer phenotype.

Article Snippet: Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking Solution (#52023; Agilent, Santa Clara, CA, USA) for 10 min. Anti-DOG1 mouse monoclonal antibody MSVA-201M (MS Validated Antibodies, Hamburg, Germany) was applied at 37 °C, 60 min, at 1:150.

Techniques: Immunostaining

Journal: PeerJ

Article Title: DOG1 is commonly expressed in pancreatic adenocarcinoma but unrelated to cancer aggressiveness

doi: 10.7717/peerj.11905

Figure Lengend Snippet: DOG1 immunostaining and CD8 positivity.

Article Snippet: Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking Solution (#52023; Agilent, Santa Clara, CA, USA) for 10 min. Anti-DOG1 mouse monoclonal antibody MSVA-201M (MS Validated Antibodies, Hamburg, Germany) was applied at 37 °C, 60 min, at 1:150.

Techniques: Immunostaining