Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: PCR primer pairs used to characterize cerebellar ANO1 and ANO2.

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Cloning

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: (A) Membrane topology model for anoctamin Ca 2+ -activated Cl - channels based on the X-ray structure of a fungal TMEM16 protein . The transmembrane domains 5 and 6 are thought to provide the pore-lining region in the homodimeric channel . Five negatively charged amino-acid residues (E , D) and an asparagine residue (N) in transmembrane domains 6–8 serve as Ca 2+ -binding sites involved in channel gating [ – ]. Four alternatively spliced segments (a—d) determine the apparent Ca 2+ -sensitivity of the ANO1 channel . ANO2 has two isoforms A and B and a regulatory motif at a position homologous to segment c in ANO1 . (B) RT-PCR analysis from mouse olfactory epithelium (OE) and mouse cerebellum (CB) yield similarly strong ANO1 signals in cerebellum but weaker signals for ANO2. (C) Immunoblots obtained from lysates of cerebellum (CB) and main olfactory epithelium (OE) from wild-type and Ano2 -/- mice show an ANO1-specific signal at ~120 kDa with the ANO1 in antiserum. (D) Rabbit anti-ANO2 ex serum stains ANO2-specific bands (asterisks) in immunoblots obtained from lysates of main olfactory epithelium (OE) and eye, as well as in membrane-protein preparations of main olfactory bulb (OB) and cerebellum (CB) . ANO2 bands are not present in immunoblots from Ano2 -/- mice.

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Membrane, Residue, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: (A) ANO2 immunosignals from the cerebellar cortex are discernible in the dendrites of Purkinje cells. The signals are weak but stronger than the background signals emanating from the granule cell layer (GL) . (B) In the Purkinje cell, the ANO2 immunosignal (green) is visible in dendrites and the perinuclear region, but only weakly in the plasma membrane of the cell body. In contrast, ANO1 signals (red) label the entire Purkinje cell soma, but are not detectable in dendrites. (C) The ANO2 antiserum does not stain the cerebellar cortex of the Ano2 -/- mouse. (D) ANO2 immunosignals in the glomeruli of the olfactory bulb serving as positive control for ANO2 in brain tissue; gl : glomerular layer, opl : outer plexiform layer. (E) Absence of ANO2 immunosignals from the olfactory bulb of the Ano2 -/- mouse. Blue in B-E represents DAPI nuclear stain. All calibrations bars: 20 μm.

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Clinical Proteomics, Membrane, Staining, Positive Control

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: (A) A cerebellar Purkinje cell loaded with the fluorescent dye Alexa Fluor 568. Scale bar: 10 μm. (B) Spontaneous postsynaptic currents in a Purkinje cell with E Cl near 0 mV and V hold = -69 mV. Overlay of 764 current traces showing similar time courses but differing amplitudes, probably reflecting distinct positions of GABAergic synapses on the Purkinje cell dendritic tree. (C) Postsynaptic currents were completely blocked by 50 μM picrotoxin, an inhibitor of GABA A -receptor chloride channels. (D) Protocol for activation of climbing fibers: Ten 0.1-ms current pulses were applied to the area near the proximal dendrite of a Purkinje cell while recording the whole-cell current of that cell at -70 mV. CF-activation produced characteristic complex spikes, as shown in the inset. (E) Upper traces : GABAergic inhibitory postsynaptic currents recorded from a Purkinje cell at V hold = -48 mV and with 5 mM Cl - in the pipette solution. The positive polarity of IPSCs indicates Cl - influx. Lower traces : postsynaptic currents, recorded immediately after the climbing-fiber stimulation, displayed decreased amplitudes. (F) IPSCs recorded from a Purkinje cell of an Ano2 -/— mouse before (upper traces) and immediately after (lower traces) CF-activation. (G) The number of detectable IPSC signals decreased by ~47% through climbing-fiber stimulation (before CF: 30.7 ± 6.5 min -1 ; after CF: 14.6 ± 3.4 min -1 ; 8 cells; ctrl ). In slices from Ano2 -/- mice, more IPSCs were detected (54.5 ± 18.5 min -1 ; 4 cells), and the activation of climbing fibers had no effect (52.5 ± 16.2 min -1 ; 4 cells).

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Activation Assay, Produced, Transferring

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: (A) Whole-cell recording from an HEK293 cell transfected with mouse cerebellar ANO2 isoform B at -70 mV. Chloride inward current was activated by diffusion of 7.5 μM Ca 2+ from the pipette into the cell immediately after whole-cell breakthrough (arrow) . (B) Results from current recordings without (black bars) and with (hatched bars) T16Ainh-A01 revealed that neither mouse ANO1 ac nor ANO1 abc were significantly inhibited by 5 μM of the compound, while the ANO2 isoform B showed a significantly reduced current density. At 25 μM T16Ainh-A01 , significant inhibition was also observed with ANO1 ac . Results were averaged from 19–30 cells for each condition. (C) When applied at the ANO2-specific concentration of 5 μM, T16Ainh-A01 blocked DDI : The number of detectable IPSCs was 48.9 ± 12.1 min -1 before and 53.4 ± 15.4 min -1 after CF stimulation. ANO2 +/+ control data from are included for comparison. (D) IPSC traces from wildtype mice in the presence of 5 μM T16Ainh-A01 show no difference in IPSC shapes and amplitudes before (left traces) and after (right traces) CF-activation.

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Transfection, Diffusion-based Assay, Transferring, Inhibition, Concentration Assay, Control, Comparison, Activation Assay

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: (A) At a Cl - concentration of 12 mM in the recording pipette, GABAergic postsynaptic currents were negative (Cl - efflux), indicating that postsynaptic E Cl is less negative than V hold . (B) Shortly after applying 5 μM ANO2 inhibitor, positive currents appear (circles) as some synapses experience a decline of postsynaptic [Cl - ] i , while others still have high Cl - (asterisks) . (C) During the continued presence of the ANO2 inhibitor, virtually all postsynaptic currents reverse to positive polarity (Cl - influx) indicating that GABAergic synapses experience an E Cl more negative than V hold . (D) The collected data from 12 Purkinje cells at [Cl - ] i = 12 mM and V hold = -60 mV demonstrate the polarity reversal of postsynaptic currents (PSCs) actuated by the ANO2 inhibitor. (E) Schematic representation of an hypothesis for the 12 mM [Cl - ] i experiment. In the absence of the ANO2 inhibitor (upper scheme) , the basal activity of ANO2 channels (green) provides a Cl - conductance in the dendritic membrane. ANO2 contributes to the Cl — transport machinery, whose various pathways are represented by the K + /Cl — cotransporter KCC2 (blue) . Together the Cl - pathways stabilize a slightly elevated level of [Cl - ] i which results in a negative driving force (V m —E Cl < 0) for Cl - currents through GABA A receptors in GABAergic synapses (red) . In this situation, Cl - currents are outwardly directed and cause negative postsynaptic currents. Application of the ANO2 inhibitor (lower scheme) reduces the Cl - conductance. This causes a polarity reversal of the Cl - driving force, as the balance shifts towards Cl - extrusion, causing local [Cl - ] i to decrease. This hypothesis provides a qualitative concept for the role of ANO2 channels in the inversion of postsynaptic currents that is depicted in panels A to C. The proximity of Cl — transport pathways and GABAergic synapses, as well as the occurrence of local Cl -. gradients within dendritic segments, are inspired by the model for GABA A -receptor-mediated Cl - gradients in extended dendritic trees proposed by Jedlicka et al. (2011) .

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Concentration Assay, Transferring, Activity Assay, Membrane

Journal: Scientific reports

Article Title: Loss of ezrin expression reduced the susceptibility to the glomerular injury in mice.

doi: 10.1038/s41598-018-22846-0

Figure Lengend Snippet: Figure 2. Immunofluorescence analysis for proteins expressed in glomerular podocytes. Immunolocalisation of ezrin, other related proteins (CLIC5, NHERF2, Rho-GDIα and podocalyxin), and marker proteins (synaptopodin and podocin) was investigated in (a) WT and (b) Vil2kd/kd mouse glomeruli (Scale bar: 20 μm).

Article Snippet: Polyclonal anti-CLIC5 antibody was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Immunofluorescence, Marker

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet: ( A ) Before unroofing. From left to right: bright-field image showing a piece of glass over a neuroblastoma cells transfected with actin labeled with mCherry-Lifeact-7 and the membrane protein TMEM16F tagged with GFP; in the center an image of the red fluorescence emitted by the labeled actin; in the right an image of the green fluorescence emitted by the TMEM16F-GFP. ( B ) After unroofing only the green signal from TMEM16F is detected (right panel) and no red fluorescence is observed (center panel and the location indicated by the white arrowhead). * (asterisks) indicate the fluorescence due to edge reflections. The green signal is detected exactly where the neuroblastoma cell was located (left panel).

Article Snippet: The membranes were incubated with the corresponding primary antibodies 1:5000 for α-Tubulin (Cat# T8203, Sigma); 1:500 for TRPC1 (Cat# ACC-101, Alomone Labs)/5 (Cat# ACC-020, Alomone Labs)/6 (Cat#ACC-120, Alomone Labs) and 1:200 for TMEM16F (Cat# ACL-016, Alomone Labs) at 4°C overnight with gentle shaking.

Techniques: Transfection, Labeling, Membrane, Fluorescence

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet: ( A–D ) Control for the presence of TRPC1, TRPC5, TRPC6, tMEM16F. ( E–H ) Raw images of the gels with the marker bands. Figure 6—figure supplement 1—source data 1. Photos of the western blots.

Article Snippet: The membranes were incubated with the corresponding primary antibodies 1:5000 for α-Tubulin (Cat# T8203, Sigma); 1:500 for TRPC1 (Cat# ACC-101, Alomone Labs)/5 (Cat# ACC-020, Alomone Labs)/6 (Cat#ACC-120, Alomone Labs) and 1:200 for TMEM16F (Cat# ACL-016, Alomone Labs) at 4°C overnight with gentle shaking.

Techniques: Control, Marker, Western Blot

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet: ( A ) Four clusters found in the dataset of force-distance (F-D) traces pulled from NG108-15 cells wild type. ( B ) The Bayesian identification of the clusters in ( A ) with the candidate membrane proteins. ( C ) Confocal images of NG108-15 cells overexpressing the construct N2b-Protein-GFP, as reported by the green fluorescence emitted by GFP. ( D ) From left to right, color density plots (blue indicating a colocalization among F-D traces of more than 80%) of the clusters superimposed to an F-D curve bearing the N2B signature (85 nm segment with a flat force below 10 pN) obtained in the sample overexpressing the candidate protein TMEM16F, TRPC1, TRPC5, and TRPC6, respectively. ( E ) Global histogram of L c of the clusters in A and the clusters with the N2B signature from cells transfected with the corresponding construct. ( F ) Position of the most likely rupture regions of the proteins according to ( E ) superimposed to the cartoon of the cryo electron microscopy (cryo-EM) structure available in the PDB.

Article Snippet: The membranes were incubated with the corresponding primary antibodies 1:5000 for α-Tubulin (Cat# T8203, Sigma); 1:500 for TRPC1 (Cat# ACC-101, Alomone Labs)/5 (Cat# ACC-020, Alomone Labs)/6 (Cat#ACC-120, Alomone Labs) and 1:200 for TMEM16F (Cat# ACL-016, Alomone Labs) at 4°C overnight with gentle shaking.

Techniques: Membrane, Construct, Fluorescence, Transfection, Cryo-Electron Microscopy, Cryo-EM Sample Prep

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet: ( A ) Global histogram of cluster NG8 and the density plot. ( B ) The peaks in ( A ) represent the region where the peaks tend to occur. ( C ) Ideal profile of TMEM16F built starting from the information that 86% of the peaks happen in the loops ( D ). ( E ) Superimposition of the real and the idealized profile: the correlation between the two generates a number that is used in the probability estimation. The more peaks correspond to loops, the higher is the cross-correlation value. ( F ) Blue dots indicate the location of force peaks in the cryo electron microscopy (cryo-EM) structure of the TMEM16F.

Article Snippet: The membranes were incubated with the corresponding primary antibodies 1:5000 for α-Tubulin (Cat# T8203, Sigma); 1:500 for TRPC1 (Cat# ACC-101, Alomone Labs)/5 (Cat# ACC-020, Alomone Labs)/6 (Cat#ACC-120, Alomone Labs) and 1:200 for TMEM16F (Cat# ACL-016, Alomone Labs) at 4°C overnight with gentle shaking.

Techniques: Cryo-Electron Microscopy, Cryo-EM Sample Prep

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet:

Article Snippet: The membranes were incubated with the corresponding primary antibodies 1:5000 for α-Tubulin (Cat# T8203, Sigma); 1:500 for TRPC1 (Cat# ACC-101, Alomone Labs)/5 (Cat# ACC-020, Alomone Labs)/6 (Cat#ACC-120, Alomone Labs) and 1:200 for TMEM16F (Cat# ACL-016, Alomone Labs) at 4°C overnight with gentle shaking.

Techniques: Plasmid Preparation, Modification, Transfection, Construct, Sequencing, Labeling, Isolation, Recombinant, Software, Staining

Journal: American journal of physiology. Cell physiology

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

doi: 10.1152/ajpcell.00295.2021

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

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

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