Journal: Cell reports

Article Title: Integrated genomic and proteomic analyses identify stimulus-dependent molecular changes associated with distinct modes of skeletal muscle atrophy

doi: 10.1016/j.celrep.2021.109971

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-alpha- tubulin (11H10) , Cell Signaling Technologies , 2125S; RRID:AB_2619646.

Techniques: Recombinant, SYBR Green Assay, Membrane, Electron Microscopy, Bicinchoninic Acid Protein Assay, Cell Based Assay, Reverse Transcription, Muscles, Gene Expression, Control, Mass Spectrometry, Software

Journal: Developmental cell

Article Title: SUPPRESSING MITOCHONDRIAL RESPIRATION IS CRITICAL FOR HYPOXIA TOLERANCE IN THE FETAL GROWTH PLATE

doi: 10.1016/j.devcel.2019.04.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti LDHA , Cell Signaling Technology , Cat# 2012, RRID:AB_2137173.

Techniques: Virus, Luciferase, Plasmid Preparation, Recombinant, Saline, Reverse Transcription, SYBR Green Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Marker, Western Blot, Stripping, In Situ, Imaging, Live Cell Imaging, Membrane, Cell Based Assay, Software

Journal: Developmental cell

Article Title: SUPPRESSING MITOCHONDRIAL RESPIRATION IS CRITICAL FOR HYPOXIA TOLERANCE IN THE FETAL GROWTH PLATE

doi: 10.1016/j.devcel.2019.04.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti Caspase-3 , Cell Signaling Technology , Cat# 9662, RRID: AB_331439.

Techniques: Virus, Luciferase, Plasmid Preparation, Recombinant, Saline, Reverse Transcription, SYBR Green Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Marker, Western Blot, Stripping, In Situ, Imaging, Live Cell Imaging, Membrane, Cell Based Assay, Software

Journal: eLife

Article Title: Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition

doi: 10.7554/eLife.44161

Figure Lengend Snippet: ( a ) and ( b ) Immunoblots from total, cytoplasmic and nuclear protein fractions show no significant difference in SWI/SNF complex members upon treatment of proteasome inhibitors (MLN2238 at 200 nM). Lamin A/C and alpha tubulin are loading controls. Blots representative of at least two biological repeats. ( c ) Treatment with proteasome inhibitor, MLN2238 at 200 nM, leads to induction of IRE1α and GRP78. However, induction of these ER stress proteins is not rescued upon re-expression of SMARCB1. ( d ) Treatment with cell cycle inhibitors nocodazole (microtubule assembly inhibitor; red) at 100 nM for G401 and 300 nM for CLF_PEDS9001_T1 or RO-3306 (CDK1 inhibitor; orange) at 10 μM for both cell lines leads to accumulation of cells in G2/M after 24 hr similar to that of MLN2238 (blue). ( e ) Nocodazole and RO-3306 treatment for 24 hr leads to activation of cleaved caspase-3 and accumulation of cyclin B1. ( e ) Treatment with cell cycle inhibitors nocodazole or RO-3306 at 72 hr leads to a maximum decrease in cell viability of 65–90% depending on the cell line and compound.

Article Snippet: Antibodies used were as follows: ARID1A (Santa Cruz; sc-373784), α-tubulin (Santa Cruz; sc-5286), β-Actin (C-4) (Santa Cruz; sc-47778), β-Actin (Cell Signaling; 8457), BAF57/SMARCE1 (Bethyl Laboratories, A300-810A), BAF60a (Santa Cruz; sc-135843), BAF155 (Cell Signaling; 11956), BAF170 (Santa Cruz; sc-166237), SMARCA4 (Santa Cruz; 17798), Cleaved Caspase-3 (Cell Signaling; 9664), c-MYC (Santa Cruz; sc-764) or c-MYC (Cell Signaling; 9402), cyclin B1 (Cell Signaling; 4135 and 4138), cyclin D1 (Santa Cruz; sc-718), GAPDH (Cell Signaling; 2118S and 97166S), GRP78 (Rockland Antibodies, Limerick, PA; 200–301 F36), IRE1-alpha (Cell Signaling; 3294), lamin A/C (Cell Signaling; 2032), PSMB5 (Abcam, Cambridge, MA; ab3330), SMARCB1/SNF5 (Bethyl A301-087A), UBE2C (Proteintech, Rosemont, IL; 66087–1).

Techniques: Western Blot, Expressing, Activation Assay

Journal: eLife

Article Title: Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition

doi: 10.7554/eLife.44161

Figure Lengend Snippet: ( a ) Volcano plot identifying genes that are required for survival in SMARCB1 deficient cancers. 204 genes were significantly upregulated when comparing the log2 fold change between SMARCB1 deficient cells and SMARCB1 re-expressed cells . We assessed how loss of these genes affected viability in 3 cell lines with loss of SMARCB1 as compared to the rest of the 482 cell lines in Project Achilles DepMap 18Q3, a genome-wide loss of function screen using CRISPR-Cas9 and calculating an effect size (e.g. differential of the 3 cell lines to 482 cell lines). A negative effect size identifies genes when deleted are required for cells for survival and the 204 genes are identified in red. Deletion of UBE2C was significantly depleted. Deletion of SMARCB1 serves as a positive control in these SMARCB1 deficient cancers as these cell lines have loss of SMARCB1. ( b ) SMARCB1 deficient lines are in the top 5% of cell lines ranked by how dependent they are on UBE2C based on Project Achilles DepMap 18Q3 dataset. Three ATRT cancer cell lines (red dots; CHLA266, CHLA06, COGAR359) were compared to 482 cell lines profiled in Project Achilles (CERES dataset 18Q3). ( c ) Gene deletion of UBE2C by CRISPR-Cas9 leads to significant viability defects in RMC and MRT cell lines as compared to either SWI/SNF wt cell line, JMSU1 (day 10), or SMARCA4 mutant cell line, A549 (day 6). Error bars shown are standard deviations from two biological replicates. * indicates a two-tailed t-test p-value<0.05 and **<0.005. ( d ) Treatment with proteasome inhibitor, MLN2238 at 200 nM, leads to upregulation of cyclin B1 and this phenotype is rescued upon SMARCB1 re-expression in both CLF_PEDS0005_T1 and CLF_PEDS9001_T1. Cyclin D1 is included as a control to ensure that the effects of proteasome inhibition are specific to cyclin B1. Blots are representative of two biological replicates. ( e ) G401 xenograft tumor growth over time by individual mouse shows that treatment effects from MLN2238 can be seen as early as 8 days from treatment initiation as compared to vehicle control. Over 26 days, tumor volumes were significantly decreased in MLN2238 treated mice. **** indicates two-way ANOVA test with p-value<0.0001. ( f ) Waterfall plot of each tumor by log2 change in tumor volume on the left y-axis and correlative percent change in tumor volume on the right y-axis following 26 days of treatment with either vehicle (black) or MLN2238 (red). * indicates a two-tailed t-test p-value<0.05. ( g ) Kaplan-Meier curves from mice with G401 xenograft tumors treated with either vehicle or MLN2238 over 61 days. * indicates a p-value of 0.0489 by log-rank (Mantel-Cox) test. 10.7554/eLife.44161.019 Figure 5—source data 1. Source data for .

Article Snippet: Antibodies used were as follows: ARID1A (Santa Cruz; sc-373784), α-tubulin (Santa Cruz; sc-5286), β-Actin (C-4) (Santa Cruz; sc-47778), β-Actin (Cell Signaling; 8457), BAF57/SMARCE1 (Bethyl Laboratories, A300-810A), BAF60a (Santa Cruz; sc-135843), BAF155 (Cell Signaling; 11956), BAF170 (Santa Cruz; sc-166237), SMARCA4 (Santa Cruz; 17798), Cleaved Caspase-3 (Cell Signaling; 9664), c-MYC (Santa Cruz; sc-764) or c-MYC (Cell Signaling; 9402), cyclin B1 (Cell Signaling; 4135 and 4138), cyclin D1 (Santa Cruz; sc-718), GAPDH (Cell Signaling; 2118S and 97166S), GRP78 (Rockland Antibodies, Limerick, PA; 200–301 F36), IRE1-alpha (Cell Signaling; 3294), lamin A/C (Cell Signaling; 2032), PSMB5 (Abcam, Cambridge, MA; ab3330), SMARCB1/SNF5 (Bethyl A301-087A), UBE2C (Proteintech, Rosemont, IL; 66087–1).

Techniques: Genome Wide, CRISPR, Positive Control, Mutagenesis, Two Tailed Test, Expressing, Control, Inhibition

Journal: eLife

Article Title: Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition

doi: 10.7554/eLife.44161

Figure Lengend Snippet: ( a ) Pulse treatment with MLN2238 (2.5 μM) in CLF_PEDS9001_T1 in the setting of re-expression of SMARCB1 leads to a decreased fold change in double Annexin V+/PI + cells. Error bars represent standard deviation from at least two biological replicates. * two-tailed t-test p-value<0.05. ( b ) Viability defects seen with pulse treatment with MLN2238 can be rescued with re-expression of SMARCB1 in SMARCB1 deficient cell lines. Error bars shown are standard deviations from two biological replicates. * indicates a two-tailed t-test p-value<0.05, **<0.005, ***<0.0005. ( c ) Pulse treatment with proteasome inhibitor, MLN2238, leads to upregulation of cyclin B1 and this phenotype is rescued upon SMARCB1 re-expression in both CLF_PEDS0005_T1 and CLF_PEDS9001_T1. Cyclin D1 is included as a control to ensure that the effects of the proteasome are specific to cyclin B1. Blots are representative of two biological replicates. ( d ) and ( e ) Primary tumor RMC cell lines (CLF_PEDS0005_T1 and CLF_PEDS9001_T) do not form tumors in vivo. 5 million cells were injected subcutaneously into Taconic immunodeficient mice and were monitored for tumor formation over 41 to 54 days. ( f ) % change in body weight of mice at day 26 as compared to day one following treatment with vehicle or with MLN2238. n.s. not significant based on a two-sided t-test p-value. ( g ) Immunoblot comparing pairs of vehicle and MLN2238 treated mice. One mouse tumor which responded to MLN2238 had activation of cleaved caspase-3 and increased cyclin B1 while one non-responder had no activation of these biomarkers.

Article Snippet: Antibodies used were as follows: ARID1A (Santa Cruz; sc-373784), α-tubulin (Santa Cruz; sc-5286), β-Actin (C-4) (Santa Cruz; sc-47778), β-Actin (Cell Signaling; 8457), BAF57/SMARCE1 (Bethyl Laboratories, A300-810A), BAF60a (Santa Cruz; sc-135843), BAF155 (Cell Signaling; 11956), BAF170 (Santa Cruz; sc-166237), SMARCA4 (Santa Cruz; 17798), Cleaved Caspase-3 (Cell Signaling; 9664), c-MYC (Santa Cruz; sc-764) or c-MYC (Cell Signaling; 9402), cyclin B1 (Cell Signaling; 4135 and 4138), cyclin D1 (Santa Cruz; sc-718), GAPDH (Cell Signaling; 2118S and 97166S), GRP78 (Rockland Antibodies, Limerick, PA; 200–301 F36), IRE1-alpha (Cell Signaling; 3294), lamin A/C (Cell Signaling; 2032), PSMB5 (Abcam, Cambridge, MA; ab3330), SMARCB1/SNF5 (Bethyl A301-087A), UBE2C (Proteintech, Rosemont, IL; 66087–1).

Techniques: Expressing, Standard Deviation, Two Tailed Test, Control, In Vivo, Injection, Western Blot, Activation Assay

Journal: eLife

Article Title: Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition

doi: 10.7554/eLife.44161

Figure Lengend Snippet:

Article Snippet: Antibodies used were as follows: ARID1A (Santa Cruz; sc-373784), α-tubulin (Santa Cruz; sc-5286), β-Actin (C-4) (Santa Cruz; sc-47778), β-Actin (Cell Signaling; 8457), BAF57/SMARCE1 (Bethyl Laboratories, A300-810A), BAF60a (Santa Cruz; sc-135843), BAF155 (Cell Signaling; 11956), BAF170 (Santa Cruz; sc-166237), SMARCA4 (Santa Cruz; 17798), Cleaved Caspase-3 (Cell Signaling; 9664), c-MYC (Santa Cruz; sc-764) or c-MYC (Cell Signaling; 9402), cyclin B1 (Cell Signaling; 4135 and 4138), cyclin D1 (Santa Cruz; sc-718), GAPDH (Cell Signaling; 2118S and 97166S), GRP78 (Rockland Antibodies, Limerick, PA; 200–301 F36), IRE1-alpha (Cell Signaling; 3294), lamin A/C (Cell Signaling; 2032), PSMB5 (Abcam, Cambridge, MA; ab3330), SMARCB1/SNF5 (Bethyl A301-087A), UBE2C (Proteintech, Rosemont, IL; 66087–1).

Techniques: Luciferase, shRNA, CRISPR, Suspension, Wilms Tumor Assay, Immunohistochemistry, RNA HS Assay, Library Quantification, Reverse Transcription, SYBR Green Assay, Staining, Cell Viability Assay, Cell Based Assay, In Vitro, In Vivo, Software

Journal: The Journal of Biological Chemistry

Article Title: Adipose tissue macrophage–derived microRNA-210-3p disrupts systemic insulin sensitivity by silencing GLUT4 in obesity

doi: 10.1016/j.jbc.2024.107328

Figure Lengend Snippet:

Article Snippet: BCA Protein Assay Kit , Thermo Fisher Scientific , #23225.

Techniques: Recombinant, Staining, Transfection, Lysis, cDNA Synthesis, Western Blot, Multiplex Assay, Luciferase, Activity Assay, Plasmid Preparation, Isolation, Reverse Transcription, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Cell Based Assay, Gel Extraction, Negative Control, Control, Software

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: Clone 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) 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: 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: 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, 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