mittx  (Alomone Labs)


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  • 93

    Structured Review

    Alomone Labs mittx
    Proposed sequence of events in the role of epithelial sodium channel (ENaC)-α in barrier protection in pneumolysin (PLY)-treated human lung microvascular endothelial cells. PLY, upon pore formation, increases Ca 2+ -influx ( 3 ), which in turn mobilizes calmodulin. Calmodulin activates <t>CaMKII,</t> which in turn phosphorylates its substrate filamin A (FLN-A) ( 15 ). Phosphorylated FLN-A promotes stress fiber formation and increases capillary permeability. Activation of NSC, by either TIP peptide (binding to ENaC-α) or <t>MitTx</t> [binding to acid-sensing ion channel 1a (ASIC1a)], abrogates PLY-mediated CaMKII activation and protects as such from PLY-induced hyperpermeability.
    Mittx, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Epithelial Sodium Channel-α Mediates the Protective Effect of the TNF-Derived TIP Peptide in Pneumolysin-Induced Endothelial Barrier Dysfunction"

    Article Title: Epithelial Sodium Channel-α Mediates the Protective Effect of the TNF-Derived TIP Peptide in Pneumolysin-Induced Endothelial Barrier Dysfunction

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00842

    Proposed sequence of events in the role of epithelial sodium channel (ENaC)-α in barrier protection in pneumolysin (PLY)-treated human lung microvascular endothelial cells. PLY, upon pore formation, increases Ca 2+ -influx ( 3 ), which in turn mobilizes calmodulin. Calmodulin activates CaMKII, which in turn phosphorylates its substrate filamin A (FLN-A) ( 15 ). Phosphorylated FLN-A promotes stress fiber formation and increases capillary permeability. Activation of NSC, by either TIP peptide (binding to ENaC-α) or MitTx [binding to acid-sensing ion channel 1a (ASIC1a)], abrogates PLY-mediated CaMKII activation and protects as such from PLY-induced hyperpermeability.
    Figure Legend Snippet: Proposed sequence of events in the role of epithelial sodium channel (ENaC)-α in barrier protection in pneumolysin (PLY)-treated human lung microvascular endothelial cells. PLY, upon pore formation, increases Ca 2+ -influx ( 3 ), which in turn mobilizes calmodulin. Calmodulin activates CaMKII, which in turn phosphorylates its substrate filamin A (FLN-A) ( 15 ). Phosphorylated FLN-A promotes stress fiber formation and increases capillary permeability. Activation of NSC, by either TIP peptide (binding to ENaC-α) or MitTx [binding to acid-sensing ion channel 1a (ASIC1a)], abrogates PLY-mediated CaMKII activation and protects as such from PLY-induced hyperpermeability.

    Techniques Used: Sequencing, Permeability, Activation Assay, Binding Assay

    2) Product Images from "Structure and analysis of nanobody binding to the human ASIC1a ion channel"

    Article Title: Structure and analysis of nanobody binding to the human ASIC1a ion channel

    Journal: eLife

    doi: 10.7554/eLife.67115

    Effects of Nb.C1 on MitTx and PcTx1 binding to hASIC1a. ( A ) Representative currents of an oocyte expressing hASIC1a activated with pH 6.0 followed by a second activation with 50 nM MitTx at pH 7.4. ( B ) Same experiment after pre-incubation of the oocyte with 50 nM Nb.C1 for 15 min. ( C ) Summary of the peak currents from pH 6.0 and MitTx activations. In this and all traces, the conditioning pH is 7.4. The bars represent the mean±SD of currents, n=8 Nb control and n=6 Nb.C1. Asterisks indicate statistical significance in t-test, p
    Figure Legend Snippet: Effects of Nb.C1 on MitTx and PcTx1 binding to hASIC1a. ( A ) Representative currents of an oocyte expressing hASIC1a activated with pH 6.0 followed by a second activation with 50 nM MitTx at pH 7.4. ( B ) Same experiment after pre-incubation of the oocyte with 50 nM Nb.C1 for 15 min. ( C ) Summary of the peak currents from pH 6.0 and MitTx activations. In this and all traces, the conditioning pH is 7.4. The bars represent the mean±SD of currents, n=8 Nb control and n=6 Nb.C1. Asterisks indicate statistical significance in t-test, p

    Techniques Used: Binding Assay, Expressing, Activation Assay, Incubation

    Structural comparison of hASIC1a-Nb.C1 complex to toxin-bound ASICs. Two side, top and bottom views of superimposed structures of hASIC1a-NbC1 complex (red) with ( A ) MitTx-bound to chicken ASIC1 (4ntw) in open conformation (orange). In side views, the threefold axis of the channel is indicated by a dashed vertical line; in top and bottom views it is indicated by dotted triangles. ( B ) PcTx1-bound chicken ASIC1 (3s3x) (gray). ( C ) Mambalgin-1-bound human ASIC1 (7ctf) (blue). Only one subunit is shown for simplicity. Surface clashes are indicated by dashed rectangles. Nb.C1, MitTx- α, MitTx- β, PcTx1, Mambalgin-1 are shown as red, orange, light-orange, light-purple, marine respectively.
    Figure Legend Snippet: Structural comparison of hASIC1a-Nb.C1 complex to toxin-bound ASICs. Two side, top and bottom views of superimposed structures of hASIC1a-NbC1 complex (red) with ( A ) MitTx-bound to chicken ASIC1 (4ntw) in open conformation (orange). In side views, the threefold axis of the channel is indicated by a dashed vertical line; in top and bottom views it is indicated by dotted triangles. ( B ) PcTx1-bound chicken ASIC1 (3s3x) (gray). ( C ) Mambalgin-1-bound human ASIC1 (7ctf) (blue). Only one subunit is shown for simplicity. Surface clashes are indicated by dashed rectangles. Nb.C1, MitTx- α, MitTx- β, PcTx1, Mambalgin-1 are shown as red, orange, light-orange, light-purple, marine respectively.

    Techniques Used:

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    Alomone Labs n m bdnf pro domain
    Differential binding of <t>pro-BDNF</t> and neurotension to sortilin and pro-sortilin. A , SPR analysis showing that unprocessed pro-BDNF (50 <t>n</t> m ) binds nearly as efficiently to the receptor in the presence (pro-sortilin) as in the absence (sortilin) of the receptor
    N M Bdnf Pro Domain, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Alomone Labs mt7
    <t>MT7</t> and pirenzepine elevated sequestration of trimeric G proteins associated with M 1 R in SH-SY5Y cells. (A) Diagrammatic representation of the experimental strategy. (B–D) Halo-tagged M 1 R was expressed transiently in sensory neurons and then treated with 100 nM MT7 or 1 μM PZ for 1 h. Cells were then lysed and Halo-M 1 R was pulled down using halo-linked resin. The halo tag was cleaved by TEV protease and the cleaved M 1 R associated multiprotein complex (MPC) was resolved in denaturing SDS-PAGE and immunoblotted using (B) anti-M 1 R, (C) anti-Gγ2/3/4/7, and (D) anti-Gα12/13 antibodies. (E,F) Scatter plot showing the relative amount of G proteins (Gα and Gγ, respectively) associated with M 1 R following drug treatment. The data represent mean ± SEM of three independent experiments. p -values ( ∗∗∗∗
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    93
    Alomone Labs mittx
    Proposed sequence of events in the role of epithelial sodium channel (ENaC)-α in barrier protection in pneumolysin (PLY)-treated human lung microvascular endothelial cells. PLY, upon pore formation, increases Ca 2+ -influx ( 3 ), which in turn mobilizes calmodulin. Calmodulin activates <t>CaMKII,</t> which in turn phosphorylates its substrate filamin A (FLN-A) ( 15 ). Phosphorylated FLN-A promotes stress fiber formation and increases capillary permeability. Activation of NSC, by either TIP peptide (binding to ENaC-α) or <t>MitTx</t> [binding to acid-sensing ion channel 1a (ASIC1a)], abrogates PLY-mediated CaMKII activation and protects as such from PLY-induced hyperpermeability.
    Mittx, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Alomone Labs qx 314
    Somatic voltage-clamp recordings from a single mitral cell, depicting responses to orthodromic stimulation of the olfactory nerve ( A ), antidromic stimulation below the mitral cell layer ( B ), and spontaneous LLDs ( E ). Antidromic and orthodromic stimulation evoke highly reproducible and nearly identical LLDs (compare A , B ). This is consistent with LLDs being generated by interactions among M/T cells. In this cell, recorded without <t>QX-314,</t> antidromically evoked LLDs are preceded by an antidromic spike ( B ). To quantify the variance of the evoked LLDs, we plotted the SDs ( SDev ) of the averaged traces ( C , D ). The largest variance occurs during the late component of the rising phase of the LLDs (delineated by the vertical dashed lines ). This is also consistent with the hypothesis that LLDs are generated by multiple interactions among M/T cells. The plot in C is shown at higher magnification in the inset . Filled circles correspond to the time points used in the text to represent the group variance data (at 20 msec after stimulus, the peak of the SD, and the peak of the LLD). F , Averaged spontaneous LLDs and their variance obtained from the traces depicted in E . Individual traces were aligned relative to the onset of their fastest component ( F, first vertical dashed line ); as in the evoked LLDs, variability is largest before the peak. G , Autocorrelogram of spontaneous LLDs ( thick line ) and peristimulus histogram ( bars ) of evoked LLDs (excluding the initial LLD) shows the similar refractory period for both spontaneous and evoked responses.
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    Image Search Results


    Differential binding of pro-BDNF and neurotension to sortilin and pro-sortilin. A , SPR analysis showing that unprocessed pro-BDNF (50 n m ) binds nearly as efficiently to the receptor in the presence (pro-sortilin) as in the absence (sortilin) of the receptor

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

    doi: 10.1074/jbc.M109.062364

    Figure Lengend Snippet: Differential binding of pro-BDNF and neurotension to sortilin and pro-sortilin. A , SPR analysis showing that unprocessed pro-BDNF (50 n m ) binds nearly as efficiently to the receptor in the presence (pro-sortilin) as in the absence (sortilin) of the receptor

    Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

    Techniques: Binding Assay, SPR Assay

    Selective competition of ligands by sortilin-derived peptide antagonist. SPR binding analysis of 50 n m unprocessed pro-BDNF ( A ), 50 n m unprocessed pro-NGF ( B ), and 90 n m RAP ( C ) to immobilized sortilin in the absence and presence of the sort166–181

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

    doi: 10.1074/jbc.M109.062364

    Figure Lengend Snippet: Selective competition of ligands by sortilin-derived peptide antagonist. SPR binding analysis of 50 n m unprocessed pro-BDNF ( A ), 50 n m unprocessed pro-NGF ( B ), and 90 n m RAP ( C ) to immobilized sortilin in the absence and presence of the sort166–181

    Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

    Techniques: Derivative Assay, SPR Assay, Binding Assay

    Mutation of the linear binding site specifically impairs binding of both the NGF and the BDNF pro-domains. SPR analysis showing reduced binding of equal amounts (analyte concentration: 200 n m ) of the soluble extracellular domains of sortilin-4A compared

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

    doi: 10.1074/jbc.M109.062364

    Figure Lengend Snippet: Mutation of the linear binding site specifically impairs binding of both the NGF and the BDNF pro-domains. SPR analysis showing reduced binding of equal amounts (analyte concentration: 200 n m ) of the soluble extracellular domains of sortilin-4A compared

    Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

    Techniques: Mutagenesis, Binding Assay, SPR Assay, Concentration Assay

    MT7 and pirenzepine elevated sequestration of trimeric G proteins associated with M 1 R in SH-SY5Y cells. (A) Diagrammatic representation of the experimental strategy. (B–D) Halo-tagged M 1 R was expressed transiently in sensory neurons and then treated with 100 nM MT7 or 1 μM PZ for 1 h. Cells were then lysed and Halo-M 1 R was pulled down using halo-linked resin. The halo tag was cleaved by TEV protease and the cleaved M 1 R associated multiprotein complex (MPC) was resolved in denaturing SDS-PAGE and immunoblotted using (B) anti-M 1 R, (C) anti-Gγ2/3/4/7, and (D) anti-Gα12/13 antibodies. (E,F) Scatter plot showing the relative amount of G proteins (Gα and Gγ, respectively) associated with M 1 R following drug treatment. The data represent mean ± SEM of three independent experiments. p -values ( ∗∗∗∗

    Journal: Frontiers in Neuroscience

    Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

    doi: 10.3389/fnins.2018.00402

    Figure Lengend Snippet: MT7 and pirenzepine elevated sequestration of trimeric G proteins associated with M 1 R in SH-SY5Y cells. (A) Diagrammatic representation of the experimental strategy. (B–D) Halo-tagged M 1 R was expressed transiently in sensory neurons and then treated with 100 nM MT7 or 1 μM PZ for 1 h. Cells were then lysed and Halo-M 1 R was pulled down using halo-linked resin. The halo tag was cleaved by TEV protease and the cleaved M 1 R associated multiprotein complex (MPC) was resolved in denaturing SDS-PAGE and immunoblotted using (B) anti-M 1 R, (C) anti-Gγ2/3/4/7, and (D) anti-Gα12/13 antibodies. (E,F) Scatter plot showing the relative amount of G proteins (Gα and Gγ, respectively) associated with M 1 R following drug treatment. The data represent mean ± SEM of three independent experiments. p -values ( ∗∗∗∗

    Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

    Techniques: SDS Page

    MT7 and pirenzepine elevated sequestration of trimeric G proteins in a protein complex associated with M 1 R in SH-SY5Y cells. (A,B) BN-PAGE analysis showing recruitment and sequestration of G proteins to M 1 R following MT7 treatment. GFP-M 1 R transfected cells were treated with 100 nM MT7 and incubated for 1 h. Cell lysates were then separated on 1D BN PAGE followed by 2D SDS-PAGE, (A) Control, (B) 100 nM MT7 treatment. Top panel: Coomassie stained gel piece showing 1D BN-PAGE separation of native page protein molecular weight marker. The red horizontal and vertical arrows indicate the direction of the 1D BN-PAGE and 2D SDS-PAGE, respectively. The blue rectangle shows PTMs of GFP-M 1 R associated with 1000 kDa and > 1200 kDa MPCs. The red circle indicates native form of the GFP-M 1 R associated with the MPCs. The red arrow connecting the red circles indicates shift of molecular weight in MPC due to recruitment of G proteins following drug treatment. The red rectangle in the bottom panel shows the co-migration of possible interacting G proteins with the MPCs.

    Journal: Frontiers in Neuroscience

    Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

    doi: 10.3389/fnins.2018.00402

    Figure Lengend Snippet: MT7 and pirenzepine elevated sequestration of trimeric G proteins in a protein complex associated with M 1 R in SH-SY5Y cells. (A,B) BN-PAGE analysis showing recruitment and sequestration of G proteins to M 1 R following MT7 treatment. GFP-M 1 R transfected cells were treated with 100 nM MT7 and incubated for 1 h. Cell lysates were then separated on 1D BN PAGE followed by 2D SDS-PAGE, (A) Control, (B) 100 nM MT7 treatment. Top panel: Coomassie stained gel piece showing 1D BN-PAGE separation of native page protein molecular weight marker. The red horizontal and vertical arrows indicate the direction of the 1D BN-PAGE and 2D SDS-PAGE, respectively. The blue rectangle shows PTMs of GFP-M 1 R associated with 1000 kDa and > 1200 kDa MPCs. The red circle indicates native form of the GFP-M 1 R associated with the MPCs. The red arrow connecting the red circles indicates shift of molecular weight in MPC due to recruitment of G proteins following drug treatment. The red rectangle in the bottom panel shows the co-migration of possible interacting G proteins with the MPCs.

    Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

    Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Incubation, SDS Page, Staining, Clear Native PAGE, Molecular Weight, Marker, Migration

    Altered mitochondrial trafficking in M 1 R overexpressing neurons was rescued by antagonist treatment. (A) Representative images (top panels) of the first frame of a series of live cell time-lapse images showing the expression of DsRed2Mito7 in the mitochondria of GFP/M 1 R–GFP expressing neurons. The circles represent mitochondria identified by MTrackJ plugin, which then tracked migration through time in a series of time lapse images and calculated velocity of specific mitochondria. White/blue line represents neurite trace. The bottom panel represents Kymographs generated from live cell time-lapse images. The Kymograph was generated using ImageJ Kymograph plugin. The X -axis represents the physical location of mitochondria on the neurite, and the Y -axis represents the location of mitochondria in time. Streak of particles traversing the kymograph from left to right in angular lines indicates retrograde/anterograde mitochondrial motion. (B) Whisker plot showing mitochondrial velocity. DRG neurons were cultured in LGF media supplemented with 100 nM MT7 or 1 μM pirenzepine for 48 h following transfection. N = 40 from three independent experiments, p -value by one-way ANOVA followed by Dunnett’s multiple comparisons test. (C) Binning of the entire data set presented in (B) . (D,E) Immunofluorescence images showing mito7-RFP and β-tubulin-III staining in the GFP/GFP-M 1 R expressing neurites. White arrows indicate continuous/discontinuous tubulin cytoskeleton in GFP/GFP-M 1 R expressing neurites, respectively. Scale bar: 5 μm.

    Journal: Frontiers in Neuroscience

    Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

    doi: 10.3389/fnins.2018.00402

    Figure Lengend Snippet: Altered mitochondrial trafficking in M 1 R overexpressing neurons was rescued by antagonist treatment. (A) Representative images (top panels) of the first frame of a series of live cell time-lapse images showing the expression of DsRed2Mito7 in the mitochondria of GFP/M 1 R–GFP expressing neurons. The circles represent mitochondria identified by MTrackJ plugin, which then tracked migration through time in a series of time lapse images and calculated velocity of specific mitochondria. White/blue line represents neurite trace. The bottom panel represents Kymographs generated from live cell time-lapse images. The Kymograph was generated using ImageJ Kymograph plugin. The X -axis represents the physical location of mitochondria on the neurite, and the Y -axis represents the location of mitochondria in time. Streak of particles traversing the kymograph from left to right in angular lines indicates retrograde/anterograde mitochondrial motion. (B) Whisker plot showing mitochondrial velocity. DRG neurons were cultured in LGF media supplemented with 100 nM MT7 or 1 μM pirenzepine for 48 h following transfection. N = 40 from three independent experiments, p -value by one-way ANOVA followed by Dunnett’s multiple comparisons test. (C) Binning of the entire data set presented in (B) . (D,E) Immunofluorescence images showing mito7-RFP and β-tubulin-III staining in the GFP/GFP-M 1 R expressing neurites. White arrows indicate continuous/discontinuous tubulin cytoskeleton in GFP/GFP-M 1 R expressing neurites, respectively. Scale bar: 5 μm.

    Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

    Techniques: Expressing, Migration, Generated, Whisker Assay, Cell Culture, Transfection, Immunofluorescence, Staining

    Restoration of cytoskeleton, mitochondrial abundance and neurite outgrowth by M 1 R antagonists MT7 and pirenzepine treatment. (A) Time-lapse live confocal images of GFP-M 1 R over-expression in sensory neurons showing MT7 induced re-localization of M 1 R from perikaryon to neurites. The overexpressed neurons were grown in defined media for 48 h and imaged (left panel). Neurons were then treated with 100 nM MT7 for 24 h and imaged (middle panel). Right panel: The same neuron depicted left was fixed and stained for β-tubulin III to show continuity of cytoskeleton. Scale bars: 50 μm. (B,C) Whiskers box (Tukey) showing total neurite outgrowth per neuron (B) and average neurite width per neuron (C) . p -value calculated by one-way ANOVA, N = 224, 230 and 198 cells, respectively, for (A) and N = 1250, 1268, 1280, and 1306 cells, respectively, for (B) . Asterisks indicate p -value calculated by unpaired t -test. (D) Whisker box plot showing amount of mitochondria in the M 1 R expressing neurons treated with 100 nM MT7 or 1 μM pirenzepine. N = 101, p -values were calculated by one-way ANOVA followed by Dunnett’s multiple comparisons tests. (E) Binning of the entire data set presented in (D) .

    Journal: Frontiers in Neuroscience

    Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

    doi: 10.3389/fnins.2018.00402

    Figure Lengend Snippet: Restoration of cytoskeleton, mitochondrial abundance and neurite outgrowth by M 1 R antagonists MT7 and pirenzepine treatment. (A) Time-lapse live confocal images of GFP-M 1 R over-expression in sensory neurons showing MT7 induced re-localization of M 1 R from perikaryon to neurites. The overexpressed neurons were grown in defined media for 48 h and imaged (left panel). Neurons were then treated with 100 nM MT7 for 24 h and imaged (middle panel). Right panel: The same neuron depicted left was fixed and stained for β-tubulin III to show continuity of cytoskeleton. Scale bars: 50 μm. (B,C) Whiskers box (Tukey) showing total neurite outgrowth per neuron (B) and average neurite width per neuron (C) . p -value calculated by one-way ANOVA, N = 224, 230 and 198 cells, respectively, for (A) and N = 1250, 1268, 1280, and 1306 cells, respectively, for (B) . Asterisks indicate p -value calculated by unpaired t -test. (D) Whisker box plot showing amount of mitochondria in the M 1 R expressing neurons treated with 100 nM MT7 or 1 μM pirenzepine. N = 101, p -values were calculated by one-way ANOVA followed by Dunnett’s multiple comparisons tests. (E) Binning of the entire data set presented in (D) .

    Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

    Techniques: Over Expression, Staining, Whisker Assay, Expressing

    M 1 R antagonists, MT7 and pirenzepine, augment neurite outgrowth in primary sensory neurons and M 1 R overexpression inhibits neurite outgrowth. (A,B) Whiskers box (Tukey) showing total neurite outgrowth per neuron. Neurons were grown for 48 h in defined media containing LGF (A) or HGF (B) condition and treated with 100 nM MT7 or 1 μM pirenzepine (PZ), respectively. N = 1249 (LGF) and 1517 (HGF), respectively. P -values were calculated by one-way ANOVA followed by post hoc multiple comparison tests. Dunnett’s multiple comparisons test was used to compare the MT7 and PZ treatment groups with the control group and Sidak’s multiple comparisons test was used to compare between the MT7 and PZ treatment groups; ∗ indicates the p -value obtained by Sidak’s multiple comparisons test. (C,D) Binning of the entire data set presented in (A,B) . (E) Immunoblot showing GFP-tagged muscarinic receptors (M 1 R to M 5 R) and GFP expression in transfected adult rat DRG neurons. pEGFP-C1-(M 1 R-M 5 R) plasmids were transfected in to DRG neurons and the lysate was resolved in SDS page and subsequently immunoblotted with anti-M 1 R (bottom panel) and anti-GFP (top panel) antibodies. (F) Time lapse confocal images showing increasing internalization (white arrows) of the GFP-M 1 R following treatment with carbachol (10 μM). Scale bar: 10 μm. (G) Immunofluorescence images showing colocalization of 24h CCh treated GFP-M1R with endosomal marker Rab5. Scale bar: 10 μm. (H) Whiskers box (Tukey) showing total neurite outgrowth per neuron, N = 634 (GFP), and N = 553 (GFP-M 1 R), neurons, respectively. P -value was calculated by t -test (unpaired). (I) Immunofluorescence images showing β-tubulin III staining and corresponding neurite trace (red lines) images in GFP and GFP-M 1 R overexpressed neurons. The total neurite outgrowth measurement was performed in Cellomics ArrayScan HCS Reader using neuronal profiling software. Scale bar: 10 μm.

    Journal: Frontiers in Neuroscience

    Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

    doi: 10.3389/fnins.2018.00402

    Figure Lengend Snippet: M 1 R antagonists, MT7 and pirenzepine, augment neurite outgrowth in primary sensory neurons and M 1 R overexpression inhibits neurite outgrowth. (A,B) Whiskers box (Tukey) showing total neurite outgrowth per neuron. Neurons were grown for 48 h in defined media containing LGF (A) or HGF (B) condition and treated with 100 nM MT7 or 1 μM pirenzepine (PZ), respectively. N = 1249 (LGF) and 1517 (HGF), respectively. P -values were calculated by one-way ANOVA followed by post hoc multiple comparison tests. Dunnett’s multiple comparisons test was used to compare the MT7 and PZ treatment groups with the control group and Sidak’s multiple comparisons test was used to compare between the MT7 and PZ treatment groups; ∗ indicates the p -value obtained by Sidak’s multiple comparisons test. (C,D) Binning of the entire data set presented in (A,B) . (E) Immunoblot showing GFP-tagged muscarinic receptors (M 1 R to M 5 R) and GFP expression in transfected adult rat DRG neurons. pEGFP-C1-(M 1 R-M 5 R) plasmids were transfected in to DRG neurons and the lysate was resolved in SDS page and subsequently immunoblotted with anti-M 1 R (bottom panel) and anti-GFP (top panel) antibodies. (F) Time lapse confocal images showing increasing internalization (white arrows) of the GFP-M 1 R following treatment with carbachol (10 μM). Scale bar: 10 μm. (G) Immunofluorescence images showing colocalization of 24h CCh treated GFP-M1R with endosomal marker Rab5. Scale bar: 10 μm. (H) Whiskers box (Tukey) showing total neurite outgrowth per neuron, N = 634 (GFP), and N = 553 (GFP-M 1 R), neurons, respectively. P -value was calculated by t -test (unpaired). (I) Immunofluorescence images showing β-tubulin III staining and corresponding neurite trace (red lines) images in GFP and GFP-M 1 R overexpressed neurons. The total neurite outgrowth measurement was performed in Cellomics ArrayScan HCS Reader using neuronal profiling software. Scale bar: 10 μm.

    Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

    Techniques: Over Expression, Expressing, Transfection, SDS Page, Immunofluorescence, Marker, Staining, Software

    Model explaining the effect of M 1 R overexpression and antagonism on tubulin associated cytoskeleton and mitochondrial trafficking. (Left) M 1 R overexpressing neurons have increased basal activity in response to secreted acetylcholine signaling through overexpressed receptor. This is turn causes recruitment of trimeric G proteins that destabilize tubulin polymers by increasing intrinsic GTPase activity of tubulin. The lack of tubulin cytoskeleton in M 1 R overexpressing neuron leads to decreased mitochondrial trafficking and stagnation of mitochondria in the neurites that impairs outgrowth. (Right) M 1 R overexpression in the presence of antagonists MT7 and pirenzepine (PZ) stabilizes tubulin polymerization. The antagonists bind to the M 1 R and may stabilize a specific structural ensemble, which in turn recruit trimeric G proteins. However, the antagonist mediated M 1 R structural ensemble may sequester the bound G proteins and makes them unavailable for exerting their effect on tubulin polymerization which in turn stabilizes microtubule cytoskeleton and promotes mitochondrial trafficking and neurite outgrowth.

    Journal: Frontiers in Neuroscience

    Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

    doi: 10.3389/fnins.2018.00402

    Figure Lengend Snippet: Model explaining the effect of M 1 R overexpression and antagonism on tubulin associated cytoskeleton and mitochondrial trafficking. (Left) M 1 R overexpressing neurons have increased basal activity in response to secreted acetylcholine signaling through overexpressed receptor. This is turn causes recruitment of trimeric G proteins that destabilize tubulin polymers by increasing intrinsic GTPase activity of tubulin. The lack of tubulin cytoskeleton in M 1 R overexpressing neuron leads to decreased mitochondrial trafficking and stagnation of mitochondria in the neurites that impairs outgrowth. (Right) M 1 R overexpression in the presence of antagonists MT7 and pirenzepine (PZ) stabilizes tubulin polymerization. The antagonists bind to the M 1 R and may stabilize a specific structural ensemble, which in turn recruit trimeric G proteins. However, the antagonist mediated M 1 R structural ensemble may sequester the bound G proteins and makes them unavailable for exerting their effect on tubulin polymerization which in turn stabilizes microtubule cytoskeleton and promotes mitochondrial trafficking and neurite outgrowth.

    Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

    Techniques: Over Expression, Activity Assay

    Knockdown of Gα13 reversed M 1 R overexpression-induced inhibition of neurite outgrowth. (A) Immunoblots showing relative expression of Gα12 and Gα13 proteins in cultured DRG neurons. (B) Scatter plot showing relative amount of Gα12 and Gα13 proteins in cultured sensory neurons. N = 5 independent experiments. p -value was calculated by unpaired t -test. (C) Immunoblots showing siRNA (cocktail of 3 siRNAs targeted to rat Gα) based knockdown of Gα13 protein in cultured adult rat DRG neurons. (D,F) Whisker box (Tukey) showing total neurite outgrowth per neuron. DRG neurons were transfected with a pEFGP-C1-M 1 R plasmid and siRNA using Amaxa nucleofection reagent and allowed to grow for 48 h. Scrambled siRNAs were used for control. In drug treatment groups, neurons were cultured in media supplemented with 100 nM MT7 or 1 μM pirenzepine following transfection. The neurons were fixed after 48 h of culture, stained with β-tubulin III and imaged using Cellomics ArrayScan HCS Reader. p -value by unpaired t -test or one-way ANOVA test followed by Dunnett’s multiple comparisons tests. N = 432/452 (in D ) and 406/694 (in F ). (E,G) Binning of the entire data set presented in (D,F) .

    Journal: Frontiers in Neuroscience

    Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

    doi: 10.3389/fnins.2018.00402

    Figure Lengend Snippet: Knockdown of Gα13 reversed M 1 R overexpression-induced inhibition of neurite outgrowth. (A) Immunoblots showing relative expression of Gα12 and Gα13 proteins in cultured DRG neurons. (B) Scatter plot showing relative amount of Gα12 and Gα13 proteins in cultured sensory neurons. N = 5 independent experiments. p -value was calculated by unpaired t -test. (C) Immunoblots showing siRNA (cocktail of 3 siRNAs targeted to rat Gα) based knockdown of Gα13 protein in cultured adult rat DRG neurons. (D,F) Whisker box (Tukey) showing total neurite outgrowth per neuron. DRG neurons were transfected with a pEFGP-C1-M 1 R plasmid and siRNA using Amaxa nucleofection reagent and allowed to grow for 48 h. Scrambled siRNAs were used for control. In drug treatment groups, neurons were cultured in media supplemented with 100 nM MT7 or 1 μM pirenzepine following transfection. The neurons were fixed after 48 h of culture, stained with β-tubulin III and imaged using Cellomics ArrayScan HCS Reader. p -value by unpaired t -test or one-way ANOVA test followed by Dunnett’s multiple comparisons tests. N = 432/452 (in D ) and 406/694 (in F ). (E,G) Binning of the entire data set presented in (D,F) .

    Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

    Techniques: Over Expression, Inhibition, Western Blot, Expressing, Cell Culture, Whisker Assay, Transfection, Plasmid Preparation, Staining

    Proposed sequence of events in the role of epithelial sodium channel (ENaC)-α in barrier protection in pneumolysin (PLY)-treated human lung microvascular endothelial cells. PLY, upon pore formation, increases Ca 2+ -influx ( 3 ), which in turn mobilizes calmodulin. Calmodulin activates CaMKII, which in turn phosphorylates its substrate filamin A (FLN-A) ( 15 ). Phosphorylated FLN-A promotes stress fiber formation and increases capillary permeability. Activation of NSC, by either TIP peptide (binding to ENaC-α) or MitTx [binding to acid-sensing ion channel 1a (ASIC1a)], abrogates PLY-mediated CaMKII activation and protects as such from PLY-induced hyperpermeability.

    Journal: Frontiers in Immunology

    Article Title: Epithelial Sodium Channel-α Mediates the Protective Effect of the TNF-Derived TIP Peptide in Pneumolysin-Induced Endothelial Barrier Dysfunction

    doi: 10.3389/fimmu.2017.00842

    Figure Lengend Snippet: Proposed sequence of events in the role of epithelial sodium channel (ENaC)-α in barrier protection in pneumolysin (PLY)-treated human lung microvascular endothelial cells. PLY, upon pore formation, increases Ca 2+ -influx ( 3 ), which in turn mobilizes calmodulin. Calmodulin activates CaMKII, which in turn phosphorylates its substrate filamin A (FLN-A) ( 15 ). Phosphorylated FLN-A promotes stress fiber formation and increases capillary permeability. Activation of NSC, by either TIP peptide (binding to ENaC-α) or MitTx [binding to acid-sensing ion channel 1a (ASIC1a)], abrogates PLY-mediated CaMKII activation and protects as such from PLY-induced hyperpermeability.

    Article Snippet: MitTx was purchased from Alomone (Jerusalem, Israel), CaMKII inhibitor XII was from EMD Millipore (Billerica, MA, USA), and the TIP peptide was custom-ordered and purchased from AMBIOPHARM (North-Augusta, SC, USA).

    Techniques: Sequencing, Permeability, Activation Assay, Binding Assay

    Somatic voltage-clamp recordings from a single mitral cell, depicting responses to orthodromic stimulation of the olfactory nerve ( A ), antidromic stimulation below the mitral cell layer ( B ), and spontaneous LLDs ( E ). Antidromic and orthodromic stimulation evoke highly reproducible and nearly identical LLDs (compare A , B ). This is consistent with LLDs being generated by interactions among M/T cells. In this cell, recorded without QX-314, antidromically evoked LLDs are preceded by an antidromic spike ( B ). To quantify the variance of the evoked LLDs, we plotted the SDs ( SDev ) of the averaged traces ( C , D ). The largest variance occurs during the late component of the rising phase of the LLDs (delineated by the vertical dashed lines ). This is also consistent with the hypothesis that LLDs are generated by multiple interactions among M/T cells. The plot in C is shown at higher magnification in the inset . Filled circles correspond to the time points used in the text to represent the group variance data (at 20 msec after stimulus, the peak of the SD, and the peak of the LLD). F , Averaged spontaneous LLDs and their variance obtained from the traces depicted in E . Individual traces were aligned relative to the onset of their fastest component ( F, first vertical dashed line ); as in the evoked LLDs, variability is largest before the peak. G , Autocorrelogram of spontaneous LLDs ( thick line ) and peristimulus histogram ( bars ) of evoked LLDs (excluding the initial LLD) shows the similar refractory period for both spontaneous and evoked responses.

    Journal: The Journal of Neuroscience

    Article Title: Long-Lasting Depolarizations in Mitral Cells of the Rat Olfactory Bulb

    doi: 10.1523/JNEUROSCI.20-05-02011.2000

    Figure Lengend Snippet: Somatic voltage-clamp recordings from a single mitral cell, depicting responses to orthodromic stimulation of the olfactory nerve ( A ), antidromic stimulation below the mitral cell layer ( B ), and spontaneous LLDs ( E ). Antidromic and orthodromic stimulation evoke highly reproducible and nearly identical LLDs (compare A , B ). This is consistent with LLDs being generated by interactions among M/T cells. In this cell, recorded without QX-314, antidromically evoked LLDs are preceded by an antidromic spike ( B ). To quantify the variance of the evoked LLDs, we plotted the SDs ( SDev ) of the averaged traces ( C , D ). The largest variance occurs during the late component of the rising phase of the LLDs (delineated by the vertical dashed lines ). This is also consistent with the hypothesis that LLDs are generated by multiple interactions among M/T cells. The plot in C is shown at higher magnification in the inset . Filled circles correspond to the time points used in the text to represent the group variance data (at 20 msec after stimulus, the peak of the SD, and the peak of the LLD). F , Averaged spontaneous LLDs and their variance obtained from the traces depicted in E . Individual traces were aligned relative to the onset of their fastest component ( F, first vertical dashed line ); as in the evoked LLDs, variability is largest before the peak. G , Autocorrelogram of spontaneous LLDs ( thick line ) and peristimulus histogram ( bars ) of evoked LLDs (excluding the initial LLD) shows the similar refractory period for both spontaneous and evoked responses.

    Article Snippet: Unless otherwise indicated, all voltage-clamp recordings were performed with QX-314 (10 m m ) in the electrode to suppress Na+ spikes.

    Techniques: Generated