anti α2δ 3  (Alomone Labs)


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  • 91
    Name:
    Anti CACNA2D3 Cavalpha2delta3 extracellular Antibody
    Description:
    Anti CACNA2D3 CaVα2δ3 extracellular Antibody ACC 103 is a highly specific antibody directed against an extracellular epitope of the rat protein The antibody can be used in western blot immunohistochemistry and immunocytochemistry applications It has been designed to recognize CaVα2δ3 from rat mouse and human samples
    Catalog Number:
    ACC-103
    Price:
    397.0
    Category:
    Primary Antibody
    Applications:
    Immunocytochemistry, Immunofluorescence, Immunohistochemistry, Live Cell Imaging, Western Blot
    Purity:
    Affinity purified on immobilized antigen.
    Immunogen:
    Synthetic peptide
    Size:
    25 mcl
    Antibody Type:
    Polyclonal Primary Antibodies
    Format:
    Lyophilized Powder
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Alomone Labs anti α2δ 3
    Anti CACNA2D3 Cavalpha2delta3 extracellular Antibody
    Anti CACNA2D3 CaVα2δ3 extracellular Antibody ACC 103 is a highly specific antibody directed against an extracellular epitope of the rat protein The antibody can be used in western blot immunohistochemistry and immunocytochemistry applications It has been designed to recognize CaVα2δ3 from rat mouse and human samples
    https://www.bioz.com/result/anti α2δ 3/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti α2δ 3 - by Bioz Stars, 2021-09
    91/100 stars

    Images

    1) Product Images from "α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels"

    Article Title: α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0511-18.2018

    Nrxn1α does not engage in stable complexes with α2δ subunits. A , IP of cotransfected α2δ subunits and Nrxn1α or control membrane proteins from HEK293 cell lysates (top). IPs of HA-tagged α2δ-1 and α2δ-3 enrich Nrxn1α::GFP (lanes 4, 5) similar to the controls neuroligin-1 (Nlgn1), E-cadherin (E-Cad) and VE-cadherin (VE-Cad; lanes 6–8). Single transfections served as control for antibody specificity (lanes 1–3). Endogenous HSP70 indicates equal amounts of lysates used (botto). B , Co-secretion of extracellular domains of α2δ-3 (α2δ-3ECD::HA) and Nrxn1α (Nrxn1α::Fc) into HEK293 cell medium with subsequent binding of the Fc moiety to protein A beads. Lysates of cells show α2δ-3ECD::HA (lanes 1–2). Whereas the positive control, Nxph1-HA, is hardly detectable in cell lysates (lane 3, bottom), it is enriched with Nrxn1α::Fc (lane 6, bottom). α2δ-3ECD::HA is enriched similarly with Nrxn1α::Fc (lane 5, top) but also with the Fc-tag alone. C , Diagram of the cleavage experiment using HRV 3C protease to release the Nrxn1αECD from Fc-beads (immunoblot data in D ). Left, Nxph1 (magenta) is bound to Nrxn1αECD (green) as expected. Right, α2δ-3ECD (cyan) remains on Fc-coupled beads (orange) but does not interact with Nrxn1αECD. D , Immunoblot of the cleavage experiment ( C ) that starts from the precipitated samples in B . After addition of protease, α2δ-3ECD remains on Fc-tag bound to beads (lanes 7, 8) but is not found on Nrxn1αECD in the supernatant (lane 11). The positive control, Nxph1, is bound to the released Nrxn1αECD (lane 12). α2δ-3 and Nph1 are shown by immunoblot, Nrxn1α and Fc proteins are visualized by UV light.
    Figure Legend Snippet: Nrxn1α does not engage in stable complexes with α2δ subunits. A , IP of cotransfected α2δ subunits and Nrxn1α or control membrane proteins from HEK293 cell lysates (top). IPs of HA-tagged α2δ-1 and α2δ-3 enrich Nrxn1α::GFP (lanes 4, 5) similar to the controls neuroligin-1 (Nlgn1), E-cadherin (E-Cad) and VE-cadherin (VE-Cad; lanes 6–8). Single transfections served as control for antibody specificity (lanes 1–3). Endogenous HSP70 indicates equal amounts of lysates used (botto). B , Co-secretion of extracellular domains of α2δ-3 (α2δ-3ECD::HA) and Nrxn1α (Nrxn1α::Fc) into HEK293 cell medium with subsequent binding of the Fc moiety to protein A beads. Lysates of cells show α2δ-3ECD::HA (lanes 1–2). Whereas the positive control, Nxph1-HA, is hardly detectable in cell lysates (lane 3, bottom), it is enriched with Nrxn1α::Fc (lane 6, bottom). α2δ-3ECD::HA is enriched similarly with Nrxn1α::Fc (lane 5, top) but also with the Fc-tag alone. C , Diagram of the cleavage experiment using HRV 3C protease to release the Nrxn1αECD from Fc-beads (immunoblot data in D ). Left, Nxph1 (magenta) is bound to Nrxn1αECD (green) as expected. Right, α2δ-3ECD (cyan) remains on Fc-coupled beads (orange) but does not interact with Nrxn1αECD. D , Immunoblot of the cleavage experiment ( C ) that starts from the precipitated samples in B . After addition of protease, α2δ-3ECD remains on Fc-tag bound to beads (lanes 7, 8) but is not found on Nrxn1αECD in the supernatant (lane 11). The positive control, Nxph1, is bound to the released Nrxn1αECD (lane 12). α2δ-3 and Nph1 are shown by immunoblot, Nrxn1α and Fc proteins are visualized by UV light.

    Techniques Used: Transfection, Binding Assay, Positive Control

    Nrxn1α in combination with α2δ-1 facilitates Ca 2+ currents through recombinant Ca V 2.1 channels. A , Representative Ca V 2.1-mediated Ca 2+ current traces recorded from heterologous tsA201 cells expressing α1 A , β3 and α2δ-1 subunits alone (black) or together with Nrxn1α (red). Step potentials as shown (right) were used to elicit Ca 2+ currents. B , I–V relationships of Ca V 2.1/β3/α2δ-1 alone (black) or in combination with Nrxn1α (red). C , Similar analysis as in B but using α2δ-3; trace in combination with Nrxn1α in blue. D , Summary of maximum current densities for cells expressing Ca V 2.1(α1 A /β3) without an α2δ (black bars), with α2δ-1 (red) or with α2δ-3 (blue), and additionally with Nrxn1α or SynCAM1 (SCAM) as indicated below bars. Data are mean ± SEM. n = number of cells as indicated in bars from at least four independent experiments. *** p
    Figure Legend Snippet: Nrxn1α in combination with α2δ-1 facilitates Ca 2+ currents through recombinant Ca V 2.1 channels. A , Representative Ca V 2.1-mediated Ca 2+ current traces recorded from heterologous tsA201 cells expressing α1 A , β3 and α2δ-1 subunits alone (black) or together with Nrxn1α (red). Step potentials as shown (right) were used to elicit Ca 2+ currents. B , I–V relationships of Ca V 2.1/β3/α2δ-1 alone (black) or in combination with Nrxn1α (red). C , Similar analysis as in B but using α2δ-3; trace in combination with Nrxn1α in blue. D , Summary of maximum current densities for cells expressing Ca V 2.1(α1 A /β3) without an α2δ (black bars), with α2δ-1 (red) or with α2δ-3 (blue), and additionally with Nrxn1α or SynCAM1 (SCAM) as indicated below bars. Data are mean ± SEM. n = number of cells as indicated in bars from at least four independent experiments. *** p

    Techniques Used: Recombinant, Expressing

    Biophysical properties of recombinant Ca V 2.1 are not altered by Nrxn1α. A , Voltage dependence of steady-state inactivation of Ca V 2.1 channels tested by a pre-pulse protocol in tsA201 cells expressing α1 A , β3, and α2δ-1 subunits alone (black) or together with Nrxn1α (red). B , Analysis as in A expressing α1 A , β3, and α2δ-3 subunits alone (black) or together with Nrxn1α (blue). C , Tail current amplitude at −40 mV after a 10 ms voltage step to the given pre-potential, recorded in tsA201 cells expressing Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). D , Slope factor of the voltage dependence of the channel activation of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.204, by unpaired t test, t (21) = 1.32. E , Half-activation voltage of the voltage dependence of activation of Ca V 2.1/α2δ-1 tail current (as given in C ) without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.812, by unpaired t test, t (21) = 0.24. F , I–V curves of tail currents of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). G , Analysis of tail current deactivation time constant at −20 mV of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.199 by unpaired t test, t (17) = 1.34. Data are mean ± SEM. N = number of cells as shown in bars or in brackets from at least four independent experiments.
    Figure Legend Snippet: Biophysical properties of recombinant Ca V 2.1 are not altered by Nrxn1α. A , Voltage dependence of steady-state inactivation of Ca V 2.1 channels tested by a pre-pulse protocol in tsA201 cells expressing α1 A , β3, and α2δ-1 subunits alone (black) or together with Nrxn1α (red). B , Analysis as in A expressing α1 A , β3, and α2δ-3 subunits alone (black) or together with Nrxn1α (blue). C , Tail current amplitude at −40 mV after a 10 ms voltage step to the given pre-potential, recorded in tsA201 cells expressing Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). D , Slope factor of the voltage dependence of the channel activation of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.204, by unpaired t test, t (21) = 1.32. E , Half-activation voltage of the voltage dependence of activation of Ca V 2.1/α2δ-1 tail current (as given in C ) without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.812, by unpaired t test, t (21) = 0.24. F , I–V curves of tail currents of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). G , Analysis of tail current deactivation time constant at −20 mV of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.199 by unpaired t test, t (17) = 1.34. Data are mean ± SEM. N = number of cells as shown in bars or in brackets from at least four independent experiments.

    Techniques Used: Recombinant, Expressing, Mass Spectrometry, Activation Assay

    α2δ-1 auxiliary subunits together with Nrxn1α facilitate presynaptic Ca 2+ influx in TKO neurons. A , Traces of Ca 2+ fluorescence changes determined from TKO neurons cotransfected with Nrxn1α and α2δ-1 subunits. Ca 2+ transients indicated by synGCaMP6f are averaged across multiple boutons in response to 1 (red), 3 (yellow), and 10 (black) APs (arrow: start of stimulation train). Inset, Initial response to a single AP on an enlarged time scale. B , Fluorescence changes of boutons as in A from TKO neurons expressing α2δ-3 subunits together with Nrxn1α. C , Summary of mean peak synGCaMP6f signals (Δ F / F o ) of Ca 2+ transients after single AP stimulation of neurons transfected with different proteins. Data are mean ± SEM. n = ROIs/neurons (in bars), differences to WT and TKO are indicated (dotted lines); significance is given compared with WT above columns (black) and compared with TKO (blue; above dashed line). *** p
    Figure Legend Snippet: α2δ-1 auxiliary subunits together with Nrxn1α facilitate presynaptic Ca 2+ influx in TKO neurons. A , Traces of Ca 2+ fluorescence changes determined from TKO neurons cotransfected with Nrxn1α and α2δ-1 subunits. Ca 2+ transients indicated by synGCaMP6f are averaged across multiple boutons in response to 1 (red), 3 (yellow), and 10 (black) APs (arrow: start of stimulation train). Inset, Initial response to a single AP on an enlarged time scale. B , Fluorescence changes of boutons as in A from TKO neurons expressing α2δ-3 subunits together with Nrxn1α. C , Summary of mean peak synGCaMP6f signals (Δ F / F o ) of Ca 2+ transients after single AP stimulation of neurons transfected with different proteins. Data are mean ± SEM. n = ROIs/neurons (in bars), differences to WT and TKO are indicated (dotted lines); significance is given compared with WT above columns (black) and compared with TKO (blue; above dashed line). *** p

    Techniques Used: Fluorescence, Expressing, Transfection

    αNrxn modulates surface mobility of α2δ-1 and α2δ-3 auxiliary subunits differentially. A , Representative immunofluorescent images of surface α2δ-1 enriched in synaptic boutons, visualized by an antibody against the HA moiety of α2δ-1::HA cotransfected with synGCaMP6f into WT neurons (top) or TKO neurons (bottom). Scale bar, 5 μm. B , Quantification of colocalization between synGCaMP6f and surface α2δ-1-positive puncta in WT and TKO. Data are mean ± SEM; n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.433) by unpaired t test. C , Labeling of the surface population of HA-tagged α2δ-1 ( C 1 ) transfected into WT neurons using an antibody specific to the HA moiety. EGFP was cotransfected to visualize neurites ( C 2 ), merged images ( C 3 ) and an overlay of all trajectories of QD-tracked single α2δ-1 molecules in a subfield as indicated ( C 4 ); sample trajectories of QD-tracked single α2δ-1 molecules ( C 5 ). Scale bars: C 1 – D 3 , 10 μm; C 4 , D 4 , 2 μm; C 5 , D 5 , 0.5 μm. D , Labeling of surface α2δ-1 as in C using TKO neurons. E , Logarithmic distribution of diffusion coefficients for α2δ-1 on axons of WT and TKO neurons, showing more trajectories of higher mobility in TKO (see §) and fewer low mobility trajectories (see #); n = trajectories/cells; error bars (SEM) shown only in outward direction. F , Median and IQR (25–75%) of diffusion coefficients of α2δ-1 shown in E . Numbers of cells from four independent experiments (in bars). * p = 0.0277, by Kruskal–Wallis test with Dunn's post-test. G , Immunofluorescent images of surface α2δ-3 in synaptic boutons as in A . Scale bar, 5 μm. H , Quantification of colocalization between synGCaMP6f and surface α2δ-3-positive puncta in WT and TKO. Data are mean ± SEM. n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.4835), by unpaired t test. I , Logarithmic distribution of diffusion coefficients as in E but for α2δ-3. With α2δ-3, more trajectories of higher mobility occurred in WT (see §), indicating a reverse effect when compared with α2δ-1 ( E ). J , Median and IQR (25–75%) of diffusion coefficients of α2δ-3 shown in I . Numbers of cells from four independent experiments (in bars). * p = 0.0347, by Kruskal–Wallis test with Dunn's post-test.
    Figure Legend Snippet: αNrxn modulates surface mobility of α2δ-1 and α2δ-3 auxiliary subunits differentially. A , Representative immunofluorescent images of surface α2δ-1 enriched in synaptic boutons, visualized by an antibody against the HA moiety of α2δ-1::HA cotransfected with synGCaMP6f into WT neurons (top) or TKO neurons (bottom). Scale bar, 5 μm. B , Quantification of colocalization between synGCaMP6f and surface α2δ-1-positive puncta in WT and TKO. Data are mean ± SEM; n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.433) by unpaired t test. C , Labeling of the surface population of HA-tagged α2δ-1 ( C 1 ) transfected into WT neurons using an antibody specific to the HA moiety. EGFP was cotransfected to visualize neurites ( C 2 ), merged images ( C 3 ) and an overlay of all trajectories of QD-tracked single α2δ-1 molecules in a subfield as indicated ( C 4 ); sample trajectories of QD-tracked single α2δ-1 molecules ( C 5 ). Scale bars: C 1 – D 3 , 10 μm; C 4 , D 4 , 2 μm; C 5 , D 5 , 0.5 μm. D , Labeling of surface α2δ-1 as in C using TKO neurons. E , Logarithmic distribution of diffusion coefficients for α2δ-1 on axons of WT and TKO neurons, showing more trajectories of higher mobility in TKO (see §) and fewer low mobility trajectories (see #); n = trajectories/cells; error bars (SEM) shown only in outward direction. F , Median and IQR (25–75%) of diffusion coefficients of α2δ-1 shown in E . Numbers of cells from four independent experiments (in bars). * p = 0.0277, by Kruskal–Wallis test with Dunn's post-test. G , Immunofluorescent images of surface α2δ-3 in synaptic boutons as in A . Scale bar, 5 μm. H , Quantification of colocalization between synGCaMP6f and surface α2δ-3-positive puncta in WT and TKO. Data are mean ± SEM. n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.4835), by unpaired t test. I , Logarithmic distribution of diffusion coefficients as in E but for α2δ-3. With α2δ-3, more trajectories of higher mobility occurred in WT (see §), indicating a reverse effect when compared with α2δ-1 ( E ). J , Median and IQR (25–75%) of diffusion coefficients of α2δ-3 shown in I . Numbers of cells from four independent experiments (in bars). * p = 0.0347, by Kruskal–Wallis test with Dunn's post-test.

    Techniques Used: Labeling, Transfection, Diffusion-based Assay

    Related Articles

    Western Blot:

    Article Title: α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels
    Article Snippet: .. To remove DNA and RNA aggregates, samples were passed six times through 1 ml syringes (30Gx1/2 inch) and subsequently subjected to Western blot analysis as described before, using the following antibodies: anti-Cav2.1 P/Q type (Synaptic Systems, catalog #152203) 1:1000, anti-α2δ-1 (Alomone Labs, catalog #ACC-015) 1:500, anti-α2δ-3 (Alomone Lab, catalog #ACC-103) 1:500, anti-Actin (Sigma-Aldirch, catalog #A50-60) 1:2000, and anti-Nrxn123 (Synaptic Systems, catalog #175003) 1:1000. ..

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  • 91
    Alomone Labs anti α2δ 3
    Nrxn1α does not engage in stable complexes with α2δ subunits. A , IP of cotransfected α2δ subunits and Nrxn1α or control membrane proteins from HEK293 cell lysates (top). IPs of HA-tagged α2δ-1 and <t>α2δ-3</t> enrich Nrxn1α::GFP (lanes 4, 5) similar to the controls neuroligin-1 (Nlgn1), E-cadherin (E-Cad) and VE-cadherin (VE-Cad; lanes 6–8). Single transfections served as control for antibody specificity (lanes 1–3). Endogenous HSP70 indicates equal amounts of lysates used (botto). B , Co-secretion of extracellular domains of α2δ-3 (α2δ-3ECD::HA) and Nrxn1α (Nrxn1α::Fc) into HEK293 cell medium with subsequent binding of the Fc moiety to protein A beads. Lysates of cells show α2δ-3ECD::HA (lanes 1–2). Whereas the positive control, Nxph1-HA, is hardly detectable in cell lysates (lane 3, bottom), it is enriched with Nrxn1α::Fc (lane 6, bottom). α2δ-3ECD::HA is enriched similarly with Nrxn1α::Fc (lane 5, top) but also with the Fc-tag alone. C , Diagram of the cleavage experiment using HRV 3C protease to release the Nrxn1αECD from Fc-beads (immunoblot data in D ). Left, Nxph1 (magenta) is bound to Nrxn1αECD (green) as expected. Right, α2δ-3ECD (cyan) remains on Fc-coupled beads (orange) but does not interact with Nrxn1αECD. D , Immunoblot of the cleavage experiment ( C ) that starts from the precipitated samples in B . After addition of protease, α2δ-3ECD remains on Fc-tag bound to beads (lanes 7, 8) but is not found on Nrxn1αECD in the supernatant (lane 11). The positive control, Nxph1, is bound to the released Nrxn1αECD (lane 12). α2δ-3 and Nph1 are shown by immunoblot, Nrxn1α and Fc proteins are visualized by UV light.
    Anti α2δ 3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti α2δ 3/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti α2δ 3 - by Bioz Stars, 2021-09
    91/100 stars
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    Nrxn1α does not engage in stable complexes with α2δ subunits. A , IP of cotransfected α2δ subunits and Nrxn1α or control membrane proteins from HEK293 cell lysates (top). IPs of HA-tagged α2δ-1 and α2δ-3 enrich Nrxn1α::GFP (lanes 4, 5) similar to the controls neuroligin-1 (Nlgn1), E-cadherin (E-Cad) and VE-cadherin (VE-Cad; lanes 6–8). Single transfections served as control for antibody specificity (lanes 1–3). Endogenous HSP70 indicates equal amounts of lysates used (botto). B , Co-secretion of extracellular domains of α2δ-3 (α2δ-3ECD::HA) and Nrxn1α (Nrxn1α::Fc) into HEK293 cell medium with subsequent binding of the Fc moiety to protein A beads. Lysates of cells show α2δ-3ECD::HA (lanes 1–2). Whereas the positive control, Nxph1-HA, is hardly detectable in cell lysates (lane 3, bottom), it is enriched with Nrxn1α::Fc (lane 6, bottom). α2δ-3ECD::HA is enriched similarly with Nrxn1α::Fc (lane 5, top) but also with the Fc-tag alone. C , Diagram of the cleavage experiment using HRV 3C protease to release the Nrxn1αECD from Fc-beads (immunoblot data in D ). Left, Nxph1 (magenta) is bound to Nrxn1αECD (green) as expected. Right, α2δ-3ECD (cyan) remains on Fc-coupled beads (orange) but does not interact with Nrxn1αECD. D , Immunoblot of the cleavage experiment ( C ) that starts from the precipitated samples in B . After addition of protease, α2δ-3ECD remains on Fc-tag bound to beads (lanes 7, 8) but is not found on Nrxn1αECD in the supernatant (lane 11). The positive control, Nxph1, is bound to the released Nrxn1αECD (lane 12). α2δ-3 and Nph1 are shown by immunoblot, Nrxn1α and Fc proteins are visualized by UV light.

    Journal: The Journal of Neuroscience

    Article Title: α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels

    doi: 10.1523/JNEUROSCI.0511-18.2018

    Figure Lengend Snippet: Nrxn1α does not engage in stable complexes with α2δ subunits. A , IP of cotransfected α2δ subunits and Nrxn1α or control membrane proteins from HEK293 cell lysates (top). IPs of HA-tagged α2δ-1 and α2δ-3 enrich Nrxn1α::GFP (lanes 4, 5) similar to the controls neuroligin-1 (Nlgn1), E-cadherin (E-Cad) and VE-cadherin (VE-Cad; lanes 6–8). Single transfections served as control for antibody specificity (lanes 1–3). Endogenous HSP70 indicates equal amounts of lysates used (botto). B , Co-secretion of extracellular domains of α2δ-3 (α2δ-3ECD::HA) and Nrxn1α (Nrxn1α::Fc) into HEK293 cell medium with subsequent binding of the Fc moiety to protein A beads. Lysates of cells show α2δ-3ECD::HA (lanes 1–2). Whereas the positive control, Nxph1-HA, is hardly detectable in cell lysates (lane 3, bottom), it is enriched with Nrxn1α::Fc (lane 6, bottom). α2δ-3ECD::HA is enriched similarly with Nrxn1α::Fc (lane 5, top) but also with the Fc-tag alone. C , Diagram of the cleavage experiment using HRV 3C protease to release the Nrxn1αECD from Fc-beads (immunoblot data in D ). Left, Nxph1 (magenta) is bound to Nrxn1αECD (green) as expected. Right, α2δ-3ECD (cyan) remains on Fc-coupled beads (orange) but does not interact with Nrxn1αECD. D , Immunoblot of the cleavage experiment ( C ) that starts from the precipitated samples in B . After addition of protease, α2δ-3ECD remains on Fc-tag bound to beads (lanes 7, 8) but is not found on Nrxn1αECD in the supernatant (lane 11). The positive control, Nxph1, is bound to the released Nrxn1αECD (lane 12). α2δ-3 and Nph1 are shown by immunoblot, Nrxn1α and Fc proteins are visualized by UV light.

    Article Snippet: To remove DNA and RNA aggregates, samples were passed six times through 1 ml syringes (30Gx1/2 inch) and subsequently subjected to Western blot analysis as described before, using the following antibodies: anti-Cav2.1 P/Q type (Synaptic Systems, catalog #152203) 1:1000, anti-α2δ-1 (Alomone Labs, catalog #ACC-015) 1:500, anti-α2δ-3 (Alomone Lab, catalog #ACC-103) 1:500, anti-Actin (Sigma-Aldirch, catalog #A50-60) 1:2000, and anti-Nrxn123 (Synaptic Systems, catalog #175003) 1:1000.

    Techniques: Transfection, Binding Assay, Positive Control

    Nrxn1α in combination with α2δ-1 facilitates Ca 2+ currents through recombinant Ca V 2.1 channels. A , Representative Ca V 2.1-mediated Ca 2+ current traces recorded from heterologous tsA201 cells expressing α1 A , β3 and α2δ-1 subunits alone (black) or together with Nrxn1α (red). Step potentials as shown (right) were used to elicit Ca 2+ currents. B , I–V relationships of Ca V 2.1/β3/α2δ-1 alone (black) or in combination with Nrxn1α (red). C , Similar analysis as in B but using α2δ-3; trace in combination with Nrxn1α in blue. D , Summary of maximum current densities for cells expressing Ca V 2.1(α1 A /β3) without an α2δ (black bars), with α2δ-1 (red) or with α2δ-3 (blue), and additionally with Nrxn1α or SynCAM1 (SCAM) as indicated below bars. Data are mean ± SEM. n = number of cells as indicated in bars from at least four independent experiments. *** p

    Journal: The Journal of Neuroscience

    Article Title: α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels

    doi: 10.1523/JNEUROSCI.0511-18.2018

    Figure Lengend Snippet: Nrxn1α in combination with α2δ-1 facilitates Ca 2+ currents through recombinant Ca V 2.1 channels. A , Representative Ca V 2.1-mediated Ca 2+ current traces recorded from heterologous tsA201 cells expressing α1 A , β3 and α2δ-1 subunits alone (black) or together with Nrxn1α (red). Step potentials as shown (right) were used to elicit Ca 2+ currents. B , I–V relationships of Ca V 2.1/β3/α2δ-1 alone (black) or in combination with Nrxn1α (red). C , Similar analysis as in B but using α2δ-3; trace in combination with Nrxn1α in blue. D , Summary of maximum current densities for cells expressing Ca V 2.1(α1 A /β3) without an α2δ (black bars), with α2δ-1 (red) or with α2δ-3 (blue), and additionally with Nrxn1α or SynCAM1 (SCAM) as indicated below bars. Data are mean ± SEM. n = number of cells as indicated in bars from at least four independent experiments. *** p

    Article Snippet: To remove DNA and RNA aggregates, samples were passed six times through 1 ml syringes (30Gx1/2 inch) and subsequently subjected to Western blot analysis as described before, using the following antibodies: anti-Cav2.1 P/Q type (Synaptic Systems, catalog #152203) 1:1000, anti-α2δ-1 (Alomone Labs, catalog #ACC-015) 1:500, anti-α2δ-3 (Alomone Lab, catalog #ACC-103) 1:500, anti-Actin (Sigma-Aldirch, catalog #A50-60) 1:2000, and anti-Nrxn123 (Synaptic Systems, catalog #175003) 1:1000.

    Techniques: Recombinant, Expressing

    Biophysical properties of recombinant Ca V 2.1 are not altered by Nrxn1α. A , Voltage dependence of steady-state inactivation of Ca V 2.1 channels tested by a pre-pulse protocol in tsA201 cells expressing α1 A , β3, and α2δ-1 subunits alone (black) or together with Nrxn1α (red). B , Analysis as in A expressing α1 A , β3, and α2δ-3 subunits alone (black) or together with Nrxn1α (blue). C , Tail current amplitude at −40 mV after a 10 ms voltage step to the given pre-potential, recorded in tsA201 cells expressing Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). D , Slope factor of the voltage dependence of the channel activation of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.204, by unpaired t test, t (21) = 1.32. E , Half-activation voltage of the voltage dependence of activation of Ca V 2.1/α2δ-1 tail current (as given in C ) without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.812, by unpaired t test, t (21) = 0.24. F , I–V curves of tail currents of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). G , Analysis of tail current deactivation time constant at −20 mV of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.199 by unpaired t test, t (17) = 1.34. Data are mean ± SEM. N = number of cells as shown in bars or in brackets from at least four independent experiments.

    Journal: The Journal of Neuroscience

    Article Title: α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels

    doi: 10.1523/JNEUROSCI.0511-18.2018

    Figure Lengend Snippet: Biophysical properties of recombinant Ca V 2.1 are not altered by Nrxn1α. A , Voltage dependence of steady-state inactivation of Ca V 2.1 channels tested by a pre-pulse protocol in tsA201 cells expressing α1 A , β3, and α2δ-1 subunits alone (black) or together with Nrxn1α (red). B , Analysis as in A expressing α1 A , β3, and α2δ-3 subunits alone (black) or together with Nrxn1α (blue). C , Tail current amplitude at −40 mV after a 10 ms voltage step to the given pre-potential, recorded in tsA201 cells expressing Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). D , Slope factor of the voltage dependence of the channel activation of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.204, by unpaired t test, t (21) = 1.32. E , Half-activation voltage of the voltage dependence of activation of Ca V 2.1/α2δ-1 tail current (as given in C ) without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.812, by unpaired t test, t (21) = 0.24. F , I–V curves of tail currents of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). G , Analysis of tail current deactivation time constant at −20 mV of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.199 by unpaired t test, t (17) = 1.34. Data are mean ± SEM. N = number of cells as shown in bars or in brackets from at least four independent experiments.

    Article Snippet: To remove DNA and RNA aggregates, samples were passed six times through 1 ml syringes (30Gx1/2 inch) and subsequently subjected to Western blot analysis as described before, using the following antibodies: anti-Cav2.1 P/Q type (Synaptic Systems, catalog #152203) 1:1000, anti-α2δ-1 (Alomone Labs, catalog #ACC-015) 1:500, anti-α2δ-3 (Alomone Lab, catalog #ACC-103) 1:500, anti-Actin (Sigma-Aldirch, catalog #A50-60) 1:2000, and anti-Nrxn123 (Synaptic Systems, catalog #175003) 1:1000.

    Techniques: Recombinant, Expressing, Mass Spectrometry, Activation Assay

    α2δ-1 auxiliary subunits together with Nrxn1α facilitate presynaptic Ca 2+ influx in TKO neurons. A , Traces of Ca 2+ fluorescence changes determined from TKO neurons cotransfected with Nrxn1α and α2δ-1 subunits. Ca 2+ transients indicated by synGCaMP6f are averaged across multiple boutons in response to 1 (red), 3 (yellow), and 10 (black) APs (arrow: start of stimulation train). Inset, Initial response to a single AP on an enlarged time scale. B , Fluorescence changes of boutons as in A from TKO neurons expressing α2δ-3 subunits together with Nrxn1α. C , Summary of mean peak synGCaMP6f signals (Δ F / F o ) of Ca 2+ transients after single AP stimulation of neurons transfected with different proteins. Data are mean ± SEM. n = ROIs/neurons (in bars), differences to WT and TKO are indicated (dotted lines); significance is given compared with WT above columns (black) and compared with TKO (blue; above dashed line). *** p

    Journal: The Journal of Neuroscience

    Article Title: α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels

    doi: 10.1523/JNEUROSCI.0511-18.2018

    Figure Lengend Snippet: α2δ-1 auxiliary subunits together with Nrxn1α facilitate presynaptic Ca 2+ influx in TKO neurons. A , Traces of Ca 2+ fluorescence changes determined from TKO neurons cotransfected with Nrxn1α and α2δ-1 subunits. Ca 2+ transients indicated by synGCaMP6f are averaged across multiple boutons in response to 1 (red), 3 (yellow), and 10 (black) APs (arrow: start of stimulation train). Inset, Initial response to a single AP on an enlarged time scale. B , Fluorescence changes of boutons as in A from TKO neurons expressing α2δ-3 subunits together with Nrxn1α. C , Summary of mean peak synGCaMP6f signals (Δ F / F o ) of Ca 2+ transients after single AP stimulation of neurons transfected with different proteins. Data are mean ± SEM. n = ROIs/neurons (in bars), differences to WT and TKO are indicated (dotted lines); significance is given compared with WT above columns (black) and compared with TKO (blue; above dashed line). *** p

    Article Snippet: To remove DNA and RNA aggregates, samples were passed six times through 1 ml syringes (30Gx1/2 inch) and subsequently subjected to Western blot analysis as described before, using the following antibodies: anti-Cav2.1 P/Q type (Synaptic Systems, catalog #152203) 1:1000, anti-α2δ-1 (Alomone Labs, catalog #ACC-015) 1:500, anti-α2δ-3 (Alomone Lab, catalog #ACC-103) 1:500, anti-Actin (Sigma-Aldirch, catalog #A50-60) 1:2000, and anti-Nrxn123 (Synaptic Systems, catalog #175003) 1:1000.

    Techniques: Fluorescence, Expressing, Transfection

    αNrxn modulates surface mobility of α2δ-1 and α2δ-3 auxiliary subunits differentially. A , Representative immunofluorescent images of surface α2δ-1 enriched in synaptic boutons, visualized by an antibody against the HA moiety of α2δ-1::HA cotransfected with synGCaMP6f into WT neurons (top) or TKO neurons (bottom). Scale bar, 5 μm. B , Quantification of colocalization between synGCaMP6f and surface α2δ-1-positive puncta in WT and TKO. Data are mean ± SEM; n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.433) by unpaired t test. C , Labeling of the surface population of HA-tagged α2δ-1 ( C 1 ) transfected into WT neurons using an antibody specific to the HA moiety. EGFP was cotransfected to visualize neurites ( C 2 ), merged images ( C 3 ) and an overlay of all trajectories of QD-tracked single α2δ-1 molecules in a subfield as indicated ( C 4 ); sample trajectories of QD-tracked single α2δ-1 molecules ( C 5 ). Scale bars: C 1 – D 3 , 10 μm; C 4 , D 4 , 2 μm; C 5 , D 5 , 0.5 μm. D , Labeling of surface α2δ-1 as in C using TKO neurons. E , Logarithmic distribution of diffusion coefficients for α2δ-1 on axons of WT and TKO neurons, showing more trajectories of higher mobility in TKO (see §) and fewer low mobility trajectories (see #); n = trajectories/cells; error bars (SEM) shown only in outward direction. F , Median and IQR (25–75%) of diffusion coefficients of α2δ-1 shown in E . Numbers of cells from four independent experiments (in bars). * p = 0.0277, by Kruskal–Wallis test with Dunn's post-test. G , Immunofluorescent images of surface α2δ-3 in synaptic boutons as in A . Scale bar, 5 μm. H , Quantification of colocalization between synGCaMP6f and surface α2δ-3-positive puncta in WT and TKO. Data are mean ± SEM. n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.4835), by unpaired t test. I , Logarithmic distribution of diffusion coefficients as in E but for α2δ-3. With α2δ-3, more trajectories of higher mobility occurred in WT (see §), indicating a reverse effect when compared with α2δ-1 ( E ). J , Median and IQR (25–75%) of diffusion coefficients of α2δ-3 shown in I . Numbers of cells from four independent experiments (in bars). * p = 0.0347, by Kruskal–Wallis test with Dunn's post-test.

    Journal: The Journal of Neuroscience

    Article Title: α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels

    doi: 10.1523/JNEUROSCI.0511-18.2018

    Figure Lengend Snippet: αNrxn modulates surface mobility of α2δ-1 and α2δ-3 auxiliary subunits differentially. A , Representative immunofluorescent images of surface α2δ-1 enriched in synaptic boutons, visualized by an antibody against the HA moiety of α2δ-1::HA cotransfected with synGCaMP6f into WT neurons (top) or TKO neurons (bottom). Scale bar, 5 μm. B , Quantification of colocalization between synGCaMP6f and surface α2δ-1-positive puncta in WT and TKO. Data are mean ± SEM; n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.433) by unpaired t test. C , Labeling of the surface population of HA-tagged α2δ-1 ( C 1 ) transfected into WT neurons using an antibody specific to the HA moiety. EGFP was cotransfected to visualize neurites ( C 2 ), merged images ( C 3 ) and an overlay of all trajectories of QD-tracked single α2δ-1 molecules in a subfield as indicated ( C 4 ); sample trajectories of QD-tracked single α2δ-1 molecules ( C 5 ). Scale bars: C 1 – D 3 , 10 μm; C 4 , D 4 , 2 μm; C 5 , D 5 , 0.5 μm. D , Labeling of surface α2δ-1 as in C using TKO neurons. E , Logarithmic distribution of diffusion coefficients for α2δ-1 on axons of WT and TKO neurons, showing more trajectories of higher mobility in TKO (see §) and fewer low mobility trajectories (see #); n = trajectories/cells; error bars (SEM) shown only in outward direction. F , Median and IQR (25–75%) of diffusion coefficients of α2δ-1 shown in E . Numbers of cells from four independent experiments (in bars). * p = 0.0277, by Kruskal–Wallis test with Dunn's post-test. G , Immunofluorescent images of surface α2δ-3 in synaptic boutons as in A . Scale bar, 5 μm. H , Quantification of colocalization between synGCaMP6f and surface α2δ-3-positive puncta in WT and TKO. Data are mean ± SEM. n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.4835), by unpaired t test. I , Logarithmic distribution of diffusion coefficients as in E but for α2δ-3. With α2δ-3, more trajectories of higher mobility occurred in WT (see §), indicating a reverse effect when compared with α2δ-1 ( E ). J , Median and IQR (25–75%) of diffusion coefficients of α2δ-3 shown in I . Numbers of cells from four independent experiments (in bars). * p = 0.0347, by Kruskal–Wallis test with Dunn's post-test.

    Article Snippet: To remove DNA and RNA aggregates, samples were passed six times through 1 ml syringes (30Gx1/2 inch) and subsequently subjected to Western blot analysis as described before, using the following antibodies: anti-Cav2.1 P/Q type (Synaptic Systems, catalog #152203) 1:1000, anti-α2δ-1 (Alomone Labs, catalog #ACC-015) 1:500, anti-α2δ-3 (Alomone Lab, catalog #ACC-103) 1:500, anti-Actin (Sigma-Aldirch, catalog #A50-60) 1:2000, and anti-Nrxn123 (Synaptic Systems, catalog #175003) 1:1000.

    Techniques: Labeling, Transfection, Diffusion-based Assay