asic1  (Alomone Labs)


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    Structured Review

    Alomone Labs asic1
    AT 2 cells contain <t>ACCN2</t> . A : PCR analysis showing expression of ACCN isoforms in cDNA prepared from AT 2 cells. Only ACCN2 <t>(ASIC1)</t> has a visible band. B : PCR reamplification showing expression of ACCN2 variant 2 (ASIC1a, lane 3 ) and lack of expression of ACCN2 variant 1 (ASIC1b, lane 2 ). NCBI, National Center for Biotechnology Information.
    Asic1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC"

    Article Title: Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00379.2016

    AT 2 cells contain ACCN2 . A : PCR analysis showing expression of ACCN isoforms in cDNA prepared from AT 2 cells. Only ACCN2 (ASIC1) has a visible band. B : PCR reamplification showing expression of ACCN2 variant 2 (ASIC1a, lane 3 ) and lack of expression of ACCN2 variant 1 (ASIC1b, lane 2 ). NCBI, National Center for Biotechnology Information.
    Figure Legend Snippet: AT 2 cells contain ACCN2 . A : PCR analysis showing expression of ACCN isoforms in cDNA prepared from AT 2 cells. Only ACCN2 (ASIC1) has a visible band. B : PCR reamplification showing expression of ACCN2 variant 2 (ASIC1a, lane 3 ) and lack of expression of ACCN2 variant 1 (ASIC1b, lane 2 ). NCBI, National Center for Biotechnology Information.

    Techniques Used: Polymerase Chain Reaction, Expressing, Variant Assay

    KO mice have no detectable ASIC1a. PCR analysis shows expression of ACCN2 in cDNA prepared from wild-type lungs and from rat lung as a positive control. The cDNA prepared from ASIC1 KO lung has no detectable ACCN2 (ASIC1).
    Figure Legend Snippet: KO mice have no detectable ASIC1a. PCR analysis shows expression of ACCN2 in cDNA prepared from wild-type lungs and from rat lung as a positive control. The cDNA prepared from ASIC1 KO lung has no detectable ACCN2 (ASIC1).

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Expressing, Positive Control

    NSC channel frequency strongly depends on ASIC1a and α-ENaC presence. A : Western blot showing ASIC1 expression in L2 cells treated with scrambled shRNA or ASIC1 silencing vectors. Although the blot is cropped, there are no other bands in the blot. Reduction of ASIC1 protein positively correlates to amount of ASIC1a shRNA used. B , left : percentage patches with NSC channels present under ASIC1 or α-ENaC knockdown conditions. Right : percentage patches with HSC channels under ASIC1 or α-ENaC knockdown conditions. Numbers above bars indicate total number of patches.
    Figure Legend Snippet: NSC channel frequency strongly depends on ASIC1a and α-ENaC presence. A : Western blot showing ASIC1 expression in L2 cells treated with scrambled shRNA or ASIC1 silencing vectors. Although the blot is cropped, there are no other bands in the blot. Reduction of ASIC1 protein positively correlates to amount of ASIC1a shRNA used. B , left : percentage patches with NSC channels present under ASIC1 or α-ENaC knockdown conditions. Right : percentage patches with HSC channels under ASIC1 or α-ENaC knockdown conditions. Numbers above bars indicate total number of patches.

    Techniques Used: Western Blot, Expressing, shRNA

    Interaction of ASIC1a and α-ENaC subunits. A , top : L2 cell protein lysate immunoprecipitated (IP) for α-ENaC and immunoblotted (IB) for ASIC1. Bottom : L2 cell protein lysate immunoprecipitated for ASIC1 and immunoblotted for α-ENaC. B : quantification of mammalian two-hybrid assay between ASIC1a and ENaC subunits. Normalized luciferase luminescence is proportional to binding affinity to ASIC1a. Negative control represents random association, and positive control represents maximum affinity. Fold increase for α-ENaC (6.6 ± 0.73) indicates a high affinity for ASIC1a. Data represent a total n of 15 ( n = 3 for each condition); * P
    Figure Legend Snippet: Interaction of ASIC1a and α-ENaC subunits. A , top : L2 cell protein lysate immunoprecipitated (IP) for α-ENaC and immunoblotted (IB) for ASIC1. Bottom : L2 cell protein lysate immunoprecipitated for ASIC1 and immunoblotted for α-ENaC. B : quantification of mammalian two-hybrid assay between ASIC1a and ENaC subunits. Normalized luciferase luminescence is proportional to binding affinity to ASIC1a. Negative control represents random association, and positive control represents maximum affinity. Fold increase for α-ENaC (6.6 ± 0.73) indicates a high affinity for ASIC1a. Data represent a total n of 15 ( n = 3 for each condition); * P

    Techniques Used: Immunoprecipitation, Two Hybrid Assay, Luciferase, Binding Assay, Negative Control, Positive Control

    ASIC1a KO increases lung water content and reduces alveolar fluid clearance. A : lung wet wt-to-dry wt ratios. Higher wet wt-to-dry wt ratio indicates increased lung water content and decreased alveolar fluid clearance. The difference in the two groups is significant. Data represent n = 8 for each treatment group. B : Evans blue dye assay showed that alveolar fluid clearance was significantly reduced in ASIC1a KO mice compared with wild-type mice. Amiloride blocked about half of AFC in wild-type mice, but there is little residual AFC after amiloride in ASIC1 KO mice. Data represent a total n = 3 mice for each treatment group; n.s., not significant. C : bronchalveolar lavage (BAL) fluid protein from wild-type and ASIC1 KO mice. There is no significant difference in BAL protein between the two groups ( n = 4 mice for each treatment group; P = 0.424).
    Figure Legend Snippet: ASIC1a KO increases lung water content and reduces alveolar fluid clearance. A : lung wet wt-to-dry wt ratios. Higher wet wt-to-dry wt ratio indicates increased lung water content and decreased alveolar fluid clearance. The difference in the two groups is significant. Data represent n = 8 for each treatment group. B : Evans blue dye assay showed that alveolar fluid clearance was significantly reduced in ASIC1a KO mice compared with wild-type mice. Amiloride blocked about half of AFC in wild-type mice, but there is little residual AFC after amiloride in ASIC1 KO mice. Data represent a total n = 3 mice for each treatment group; n.s., not significant. C : bronchalveolar lavage (BAL) fluid protein from wild-type and ASIC1 KO mice. There is no significant difference in BAL protein between the two groups ( n = 4 mice for each treatment group; P = 0.424).

    Techniques Used: Mouse Assay

    NSC channels are not observable in single-channel patches on lung slices from ASIC1 KO mice. Single-channel recordings were measured from AT 2 cells in lung slices. A : single-channel currents ( right ) and distribution of current amplitudes ( left ) in a patch on an AT 2 cell from a wild-type lung slice, which has both NSC and HSC channels (sometimes overlapping). B : single-channel currents ( right ) and distribution of current amplitudes ( left ) in a patch on an AT 2 cell from an ASIC1 KO lung slice, which has only HSC channels.
    Figure Legend Snippet: NSC channels are not observable in single-channel patches on lung slices from ASIC1 KO mice. Single-channel recordings were measured from AT 2 cells in lung slices. A : single-channel currents ( right ) and distribution of current amplitudes ( left ) in a patch on an AT 2 cell from a wild-type lung slice, which has both NSC and HSC channels (sometimes overlapping). B : single-channel currents ( right ) and distribution of current amplitudes ( left ) in a patch on an AT 2 cell from an ASIC1 KO lung slice, which has only HSC channels.

    Techniques Used: Mouse Assay

    NSC channels are sensitive to ASIC1-modifying toxins. A : NSC channels were activated by venom of the Texas coral snake ( Micrurus tener tener ). B : psalmotoxin-1 isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula), is a potent and selective acid-sensing ion channel 1a (ASIC1a) blocker (IC 50 ). When applied to AT 2 cells in primary culture, it uniformly decreased NSC open probability. Neither toxin had any effect on HSC channels. * P
    Figure Legend Snippet: NSC channels are sensitive to ASIC1-modifying toxins. A : NSC channels were activated by venom of the Texas coral snake ( Micrurus tener tener ). B : psalmotoxin-1 isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula), is a potent and selective acid-sensing ion channel 1a (ASIC1a) blocker (IC 50 ). When applied to AT 2 cells in primary culture, it uniformly decreased NSC open probability. Neither toxin had any effect on HSC channels. * P

    Techniques Used: Isolation

    2) Product Images from "ASIC1a senses lactate uptake to regulate metabolism in neurons"

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    Journal: Redox Biology

    doi: 10.1016/j.redox.2022.102253

    ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p
    Figure Legend Snippet: ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p

    Techniques Used: Fluorescence

    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p
    Figure Legend Snippet: Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p

    Techniques Used: Cell Culture, Fluorescence

    3) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    4) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

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    Alomone Labs psalmotoxin 1 pctx1
    Effect of amiloride, mambalgin-1, APETx2 and <t>PcTx1</t> on ASIC1b-expressing muscle afferent DRG neurons. (A) Whole-cell patch clamp recording on an ASIC1b-expressing DRG neuron projecting to gastrocnemius muscle labeled by fluorogold. (B) Mambalgin-1 (MB-1) (1 μM) inhibited acid (pH 5.0)-induced currents in 13 of 14 ASIC1b-expressing muscle afferent DRG neurons. (C) APETx2 (1 μM) inhibited acid (pH 5.0)-induced currents in 6 of 13 ASIC1b-expressing muscle afferent DRG neurons. (D) PcTx1 (100 nM) inhibited acid (pH 5.0)-induced currents in 5 of 11 ASIC1b-expressing muscle afferent DRG neurons.
    Psalmotoxin 1 Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of amiloride, mambalgin-1, APETx2 and PcTx1 on ASIC1b-expressing muscle afferent DRG neurons. (A) Whole-cell patch clamp recording on an ASIC1b-expressing DRG neuron projecting to gastrocnemius muscle labeled by fluorogold. (B) Mambalgin-1 (MB-1) (1 μM) inhibited acid (pH 5.0)-induced currents in 13 of 14 ASIC1b-expressing muscle afferent DRG neurons. (C) APETx2 (1 μM) inhibited acid (pH 5.0)-induced currents in 6 of 13 ASIC1b-expressing muscle afferent DRG neurons. (D) PcTx1 (100 nM) inhibited acid (pH 5.0)-induced currents in 5 of 11 ASIC1b-expressing muscle afferent DRG neurons.

    Journal: Frontiers in Neuroscience

    Article Title: Involvement of Acid-Sensing Ion Channel 1b in the Development of Acid-Induced Chronic Muscle Pain

    doi: 10.3389/fnins.2019.01247

    Figure Lengend Snippet: Effect of amiloride, mambalgin-1, APETx2 and PcTx1 on ASIC1b-expressing muscle afferent DRG neurons. (A) Whole-cell patch clamp recording on an ASIC1b-expressing DRG neuron projecting to gastrocnemius muscle labeled by fluorogold. (B) Mambalgin-1 (MB-1) (1 μM) inhibited acid (pH 5.0)-induced currents in 13 of 14 ASIC1b-expressing muscle afferent DRG neurons. (C) APETx2 (1 μM) inhibited acid (pH 5.0)-induced currents in 6 of 13 ASIC1b-expressing muscle afferent DRG neurons. (D) PcTx1 (100 nM) inhibited acid (pH 5.0)-induced currents in 5 of 11 ASIC1b-expressing muscle afferent DRG neurons.

    Article Snippet: Mambalgin-1, APETx2, and psalmotoxin 1 (PcTx1) were purchased from Alomone Labs and prepared by autoclaved water in stock solutions of 200 μM, 1 mM, and 200 μM respectively.

    Techniques: Expressing, Patch Clamp, Labeling

    AT 2 cells contain ACCN2 . A : PCR analysis showing expression of ACCN isoforms in cDNA prepared from AT 2 cells. Only ACCN2 (ASIC1) has a visible band. B : PCR reamplification showing expression of ACCN2 variant 2 (ASIC1a, lane 3 ) and lack of expression of ACCN2 variant 1 (ASIC1b, lane 2 ). NCBI, National Center for Biotechnology Information.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC

    doi: 10.1152/ajplung.00379.2016

    Figure Lengend Snippet: AT 2 cells contain ACCN2 . A : PCR analysis showing expression of ACCN isoforms in cDNA prepared from AT 2 cells. Only ACCN2 (ASIC1) has a visible band. B : PCR reamplification showing expression of ACCN2 variant 2 (ASIC1a, lane 3 ) and lack of expression of ACCN2 variant 1 (ASIC1b, lane 2 ). NCBI, National Center for Biotechnology Information.

    Article Snippet: It is a potent, persistent, and selective agonist for acid-sensing ion channels and is highly selective for ASIC1 at neutral pH ( , Psalmotoxin-1 is isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula).

    Techniques: Polymerase Chain Reaction, Expressing, Variant Assay

    KO mice have no detectable ASIC1a. PCR analysis shows expression of ACCN2 in cDNA prepared from wild-type lungs and from rat lung as a positive control. The cDNA prepared from ASIC1 KO lung has no detectable ACCN2 (ASIC1).

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC

    doi: 10.1152/ajplung.00379.2016

    Figure Lengend Snippet: KO mice have no detectable ASIC1a. PCR analysis shows expression of ACCN2 in cDNA prepared from wild-type lungs and from rat lung as a positive control. The cDNA prepared from ASIC1 KO lung has no detectable ACCN2 (ASIC1).

    Article Snippet: It is a potent, persistent, and selective agonist for acid-sensing ion channels and is highly selective for ASIC1 at neutral pH ( , Psalmotoxin-1 is isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula).

    Techniques: Mouse Assay, Polymerase Chain Reaction, Expressing, Positive Control

    NSC channel frequency strongly depends on ASIC1a and α-ENaC presence. A : Western blot showing ASIC1 expression in L2 cells treated with scrambled shRNA or ASIC1 silencing vectors. Although the blot is cropped, there are no other bands in the blot. Reduction of ASIC1 protein positively correlates to amount of ASIC1a shRNA used. B , left : percentage patches with NSC channels present under ASIC1 or α-ENaC knockdown conditions. Right : percentage patches with HSC channels under ASIC1 or α-ENaC knockdown conditions. Numbers above bars indicate total number of patches.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC

    doi: 10.1152/ajplung.00379.2016

    Figure Lengend Snippet: NSC channel frequency strongly depends on ASIC1a and α-ENaC presence. A : Western blot showing ASIC1 expression in L2 cells treated with scrambled shRNA or ASIC1 silencing vectors. Although the blot is cropped, there are no other bands in the blot. Reduction of ASIC1 protein positively correlates to amount of ASIC1a shRNA used. B , left : percentage patches with NSC channels present under ASIC1 or α-ENaC knockdown conditions. Right : percentage patches with HSC channels under ASIC1 or α-ENaC knockdown conditions. Numbers above bars indicate total number of patches.

    Article Snippet: It is a potent, persistent, and selective agonist for acid-sensing ion channels and is highly selective for ASIC1 at neutral pH ( , Psalmotoxin-1 is isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula).

    Techniques: Western Blot, Expressing, shRNA

    Interaction of ASIC1a and α-ENaC subunits. A , top : L2 cell protein lysate immunoprecipitated (IP) for α-ENaC and immunoblotted (IB) for ASIC1. Bottom : L2 cell protein lysate immunoprecipitated for ASIC1 and immunoblotted for α-ENaC. B : quantification of mammalian two-hybrid assay between ASIC1a and ENaC subunits. Normalized luciferase luminescence is proportional to binding affinity to ASIC1a. Negative control represents random association, and positive control represents maximum affinity. Fold increase for α-ENaC (6.6 ± 0.73) indicates a high affinity for ASIC1a. Data represent a total n of 15 ( n = 3 for each condition); * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC

    doi: 10.1152/ajplung.00379.2016

    Figure Lengend Snippet: Interaction of ASIC1a and α-ENaC subunits. A , top : L2 cell protein lysate immunoprecipitated (IP) for α-ENaC and immunoblotted (IB) for ASIC1. Bottom : L2 cell protein lysate immunoprecipitated for ASIC1 and immunoblotted for α-ENaC. B : quantification of mammalian two-hybrid assay between ASIC1a and ENaC subunits. Normalized luciferase luminescence is proportional to binding affinity to ASIC1a. Negative control represents random association, and positive control represents maximum affinity. Fold increase for α-ENaC (6.6 ± 0.73) indicates a high affinity for ASIC1a. Data represent a total n of 15 ( n = 3 for each condition); * P

    Article Snippet: It is a potent, persistent, and selective agonist for acid-sensing ion channels and is highly selective for ASIC1 at neutral pH ( , Psalmotoxin-1 is isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula).

    Techniques: Immunoprecipitation, Two Hybrid Assay, Luciferase, Binding Assay, Negative Control, Positive Control

    ASIC1a KO increases lung water content and reduces alveolar fluid clearance. A : lung wet wt-to-dry wt ratios. Higher wet wt-to-dry wt ratio indicates increased lung water content and decreased alveolar fluid clearance. The difference in the two groups is significant. Data represent n = 8 for each treatment group. B : Evans blue dye assay showed that alveolar fluid clearance was significantly reduced in ASIC1a KO mice compared with wild-type mice. Amiloride blocked about half of AFC in wild-type mice, but there is little residual AFC after amiloride in ASIC1 KO mice. Data represent a total n = 3 mice for each treatment group; n.s., not significant. C : bronchalveolar lavage (BAL) fluid protein from wild-type and ASIC1 KO mice. There is no significant difference in BAL protein between the two groups ( n = 4 mice for each treatment group; P = 0.424).

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC

    doi: 10.1152/ajplung.00379.2016

    Figure Lengend Snippet: ASIC1a KO increases lung water content and reduces alveolar fluid clearance. A : lung wet wt-to-dry wt ratios. Higher wet wt-to-dry wt ratio indicates increased lung water content and decreased alveolar fluid clearance. The difference in the two groups is significant. Data represent n = 8 for each treatment group. B : Evans blue dye assay showed that alveolar fluid clearance was significantly reduced in ASIC1a KO mice compared with wild-type mice. Amiloride blocked about half of AFC in wild-type mice, but there is little residual AFC after amiloride in ASIC1 KO mice. Data represent a total n = 3 mice for each treatment group; n.s., not significant. C : bronchalveolar lavage (BAL) fluid protein from wild-type and ASIC1 KO mice. There is no significant difference in BAL protein between the two groups ( n = 4 mice for each treatment group; P = 0.424).

    Article Snippet: It is a potent, persistent, and selective agonist for acid-sensing ion channels and is highly selective for ASIC1 at neutral pH ( , Psalmotoxin-1 is isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula).

    Techniques: Mouse Assay

    NSC channels are not observable in single-channel patches on lung slices from ASIC1 KO mice. Single-channel recordings were measured from AT 2 cells in lung slices. A : single-channel currents ( right ) and distribution of current amplitudes ( left ) in a patch on an AT 2 cell from a wild-type lung slice, which has both NSC and HSC channels (sometimes overlapping). B : single-channel currents ( right ) and distribution of current amplitudes ( left ) in a patch on an AT 2 cell from an ASIC1 KO lung slice, which has only HSC channels.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC

    doi: 10.1152/ajplung.00379.2016

    Figure Lengend Snippet: NSC channels are not observable in single-channel patches on lung slices from ASIC1 KO mice. Single-channel recordings were measured from AT 2 cells in lung slices. A : single-channel currents ( right ) and distribution of current amplitudes ( left ) in a patch on an AT 2 cell from a wild-type lung slice, which has both NSC and HSC channels (sometimes overlapping). B : single-channel currents ( right ) and distribution of current amplitudes ( left ) in a patch on an AT 2 cell from an ASIC1 KO lung slice, which has only HSC channels.

    Article Snippet: It is a potent, persistent, and selective agonist for acid-sensing ion channels and is highly selective for ASIC1 at neutral pH ( , Psalmotoxin-1 is isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula).

    Techniques: Mouse Assay

    NSC channels are sensitive to ASIC1-modifying toxins. A : NSC channels were activated by venom of the Texas coral snake ( Micrurus tener tener ). B : psalmotoxin-1 isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula), is a potent and selective acid-sensing ion channel 1a (ASIC1a) blocker (IC 50 ). When applied to AT 2 cells in primary culture, it uniformly decreased NSC open probability. Neither toxin had any effect on HSC channels. * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC

    doi: 10.1152/ajplung.00379.2016

    Figure Lengend Snippet: NSC channels are sensitive to ASIC1-modifying toxins. A : NSC channels were activated by venom of the Texas coral snake ( Micrurus tener tener ). B : psalmotoxin-1 isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula), is a potent and selective acid-sensing ion channel 1a (ASIC1a) blocker (IC 50 ). When applied to AT 2 cells in primary culture, it uniformly decreased NSC open probability. Neither toxin had any effect on HSC channels. * P

    Article Snippet: It is a potent, persistent, and selective agonist for acid-sensing ion channels and is highly selective for ASIC1 at neutral pH ( , Psalmotoxin-1 is isolated from the venom of the spider, Psalmopoeus cambridgei (Trinidad chevron tarantula).

    Techniques: Isolation

    ASIC1a channels presynaptically control transmitter release in motor nerve terminals from female mice. A : average spontaneous miniature end-plate potential (MEPP) frequencies obtained from female ( left ) and male ( right ) wild-type (filled black circles), wild type + psalmotoxin-1 (10 nM, open black circles), and ASIC1a −/− (filled gray circles) levator auris longus muscle motor nerve terminals (MNTs). In female mice, wild-type frequencies were significantly lower compared with both wild type + psalmotoxin-1 (10 nM) and ASIC1a −/− [1-way ANOVA, F (2,61) = 23.1; Bonferroni post hoc test, P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Acid-sensing ion channels 1a (ASIC1a) inhibit neuromuscular transmission in female mice

    doi: 10.1152/ajpcell.00301.2013

    Figure Lengend Snippet: ASIC1a channels presynaptically control transmitter release in motor nerve terminals from female mice. A : average spontaneous miniature end-plate potential (MEPP) frequencies obtained from female ( left ) and male ( right ) wild-type (filled black circles), wild type + psalmotoxin-1 (10 nM, open black circles), and ASIC1a −/− (filled gray circles) levator auris longus muscle motor nerve terminals (MNTs). In female mice, wild-type frequencies were significantly lower compared with both wild type + psalmotoxin-1 (10 nM) and ASIC1a −/− [1-way ANOVA, F (2,61) = 23.1; Bonferroni post hoc test, P

    Article Snippet: Toxin and chemicals.Alpha-bungarotoxin and all salts of analytical grade were purchased from Sigma (St. Louis, MO), and μ-conotoxin GIIIB and psalmotoxin-1 were purchased from Alomone Labs (Jerusamen, Israel).

    Techniques: Mouse Assay

    ASIC1a channels reduced neuromuscular transmission during 75 Hz/5 s stimulation train in motor nerve terminals of female mice. A : representative intracellular recordings of EPP are shown during nerve stimulation at 75 Hz (5 s long, 375 stimuli) using 0.8 mM HEPES/2 mM [Ca 2+ ]/1 mM [Mg 2+ ] + 10 μM μ-conotoxin GIIIB saline solution. EPP facilitate during the first stimuli and then undergo depression until a steady state was reached (top trace, wild type). Interestingly, EPP from ASIC1a −/− female mice (mid trace) and female wild type + psalmotoxin 1 (10 nM; bottom trace) presented greater initial facilitation compared with those from wild-type female mice (top trace). B : average EPP amplitude ratio [( n th stimuli amplitude/1st stimulus amplitude) × 100] of the initial 50 stimuli shown in A for levator auris longus MNTs from wild type (filled circles, n = 8 MNTs), ASIC1a −/− (open circles, n = 6 MNTs), and wild type + psalmotoxin-1 (10 nM; filled triangles, n = 5 MNTs). Wild-type ratios were significantly smaller than both wild-type + psalmotoxin-1 (10 nM) and ASIC1a −/− ones [one-way ANOVA, F (2,87) = 4.3; wild type vs. ASIC1a −/− , wild type + psalmotoxin-1 (10 nM), Bonferroni post hoc test, P = 0.016]. Wild-type + psalmotoxin-1 (10 nM) and ASIC1a −/− ratios were not significantly different (Bonferroni post hoc test, P = 1.0). * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Acid-sensing ion channels 1a (ASIC1a) inhibit neuromuscular transmission in female mice

    doi: 10.1152/ajpcell.00301.2013

    Figure Lengend Snippet: ASIC1a channels reduced neuromuscular transmission during 75 Hz/5 s stimulation train in motor nerve terminals of female mice. A : representative intracellular recordings of EPP are shown during nerve stimulation at 75 Hz (5 s long, 375 stimuli) using 0.8 mM HEPES/2 mM [Ca 2+ ]/1 mM [Mg 2+ ] + 10 μM μ-conotoxin GIIIB saline solution. EPP facilitate during the first stimuli and then undergo depression until a steady state was reached (top trace, wild type). Interestingly, EPP from ASIC1a −/− female mice (mid trace) and female wild type + psalmotoxin 1 (10 nM; bottom trace) presented greater initial facilitation compared with those from wild-type female mice (top trace). B : average EPP amplitude ratio [( n th stimuli amplitude/1st stimulus amplitude) × 100] of the initial 50 stimuli shown in A for levator auris longus MNTs from wild type (filled circles, n = 8 MNTs), ASIC1a −/− (open circles, n = 6 MNTs), and wild type + psalmotoxin-1 (10 nM; filled triangles, n = 5 MNTs). Wild-type ratios were significantly smaller than both wild-type + psalmotoxin-1 (10 nM) and ASIC1a −/− ones [one-way ANOVA, F (2,87) = 4.3; wild type vs. ASIC1a −/− , wild type + psalmotoxin-1 (10 nM), Bonferroni post hoc test, P = 0.016]. Wild-type + psalmotoxin-1 (10 nM) and ASIC1a −/− ratios were not significantly different (Bonferroni post hoc test, P = 1.0). * P

    Article Snippet: Toxin and chemicals.Alpha-bungarotoxin and all salts of analytical grade were purchased from Sigma (St. Louis, MO), and μ-conotoxin GIIIB and psalmotoxin-1 were purchased from Alomone Labs (Jerusamen, Israel).

    Techniques: Transmission Assay, Mouse Assay

    ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p

    Journal: Redox Biology

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    doi: 10.1016/j.redox.2022.102253

    Figure Lengend Snippet: ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p

    Article Snippet: 4.4 Fluorescence imaging Experiments done on WT neurons, untreated or treated with PcTX1 (Alomone labs, #STP200), were performed using the imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator (TILL Photonics, Planegg, Germany) and a SensiCam cooled charge-coupled device (PCO, Kelheim, Germany).

    Techniques: Fluorescence

    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p

    Journal: Redox Biology

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    doi: 10.1016/j.redox.2022.102253

    Figure Lengend Snippet: Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p

    Article Snippet: 4.4 Fluorescence imaging Experiments done on WT neurons, untreated or treated with PcTX1 (Alomone labs, #STP200), were performed using the imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator (TILL Photonics, Planegg, Germany) and a SensiCam cooled charge-coupled device (PCO, Kelheim, Germany).

    Techniques: Cell Culture, Fluorescence