goat anti kv3 4 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs goat anti kv3 4 antibody
    Quantification of <t>Kv3.4</t> plasma membrane immunostaining from 2 weeks after laminectomy and SCI. A , Representative immunofluorescence sections stained with anti-Kv3.4 and pan-cadherin antibodies from laminectomy ( N = 3) and SCI ( N = 3) animals. B , Histogram
    Goat Anti Kv3 4 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    goat anti kv3 4 antibody - by Bioz Stars, 2021-12
    93/100 stars

    Images

    1) Product Images from "Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury"

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Quantification of Kv3.4 plasma membrane immunostaining from 2 weeks after laminectomy and SCI. A , Representative immunofluorescence sections stained with anti-Kv3.4 and pan-cadherin antibodies from laminectomy ( N = 3) and SCI ( N = 3) animals. B , Histogram
    Figure Legend Snippet: Quantification of Kv3.4 plasma membrane immunostaining from 2 weeks after laminectomy and SCI. A , Representative immunofluorescence sections stained with anti-Kv3.4 and pan-cadherin antibodies from laminectomy ( N = 3) and SCI ( N = 3) animals. B , Histogram

    Techniques Used: Immunostaining, Immunofluorescence, Staining

    Kv3.4 current expression and inactivation from naive, laminectomy, and SCI. A , Scatter plot of I p for all data on a logarithmic scale. Dashed gray line is 1 SD from the center of the naive distribution (16.1 pA). B , I p / I 500 ratio for all groups examined.
    Figure Legend Snippet: Kv3.4 current expression and inactivation from naive, laminectomy, and SCI. A , Scatter plot of I p for all data on a logarithmic scale. Dashed gray line is 1 SD from the center of the naive distribution (16.1 pA). B , I p / I 500 ratio for all groups examined.

    Techniques Used: Expressing

    Kv3.4 current phenotypes after laminectomy and SCI. A , B , Representative Kv3.4 currents from laminectomy (Lam) and SCI neurons at 1 week and 2 weeks after surgery. Pulse protocol and scale bars are as in A . C , Pie charts showing the percentages
    Figure Legend Snippet: Kv3.4 current phenotypes after laminectomy and SCI. A , B , Representative Kv3.4 currents from laminectomy (Lam) and SCI neurons at 1 week and 2 weeks after surgery. Pulse protocol and scale bars are as in A . C , Pie charts showing the percentages

    Techniques Used: Laser Capture Microdissection

    Kv3.4 channel expression and current phenotypes in DRG neurons. A , DRG section from a naive animal immunostained as indicated. B , Distribution of peak currents from naive neurons and best fit Gaussian distribution (solid line; inset: x c = 33.8 pA, w =
    Figure Legend Snippet: Kv3.4 channel expression and current phenotypes in DRG neurons. A , DRG section from a naive animal immunostained as indicated. B , Distribution of peak currents from naive neurons and best fit Gaussian distribution (solid line; inset: x c = 33.8 pA, w =

    Techniques Used: Expressing

    Dynamic clamping of a spiking DRG neuron in the SCI group. A , Cell-attached Kv3.4 currents evoked by 150 pulses to +100 mV (black traces) and the mean current (red trace). Pulse protocol details in legend. Dashed line represents 0 pA. B , Variance
    Figure Legend Snippet: Dynamic clamping of a spiking DRG neuron in the SCI group. A , Cell-attached Kv3.4 currents evoked by 150 pulses to +100 mV (black traces) and the mean current (red trace). Pulse protocol details in legend. Dashed line represents 0 pA. B , Variance

    Techniques Used:

    Early changes: laminectomy and SCI alter the inactivation properties and expression of Kv3.4 channels in DRG neurons
    Figure Legend Snippet: Early changes: laminectomy and SCI alter the inactivation properties and expression of Kv3.4 channels in DRG neurons

    Techniques Used: Expressing

    Expression of Kv3.4 mRNA and protein from naive and 2 weeks after laminectomy and SCI. A–C , Box plots of single-cell mRNA copies of Kv3.4 mRNA (black = naive; blue = laminectomy; red = SCI). Box is 25–75 percentiles with median marked
    Figure Legend Snippet: Expression of Kv3.4 mRNA and protein from naive and 2 weeks after laminectomy and SCI. A–C , Box plots of single-cell mRNA copies of Kv3.4 mRNA (black = naive; blue = laminectomy; red = SCI). Box is 25–75 percentiles with median marked

    Techniques Used: Expressing

    Kv3.4 DRG immunostaining from naive and 2 weeks laminectomy and SCI. A – C , Representative immunofluorescence sections stained with anti-Kv3.4 antibody from naive, laminectomy and SCI animals. White arrows show neurons with significant staining
    Figure Legend Snippet: Kv3.4 DRG immunostaining from naive and 2 weeks laminectomy and SCI. A – C , Representative immunofluorescence sections stained with anti-Kv3.4 antibody from naive, laminectomy and SCI animals. White arrows show neurons with significant staining

    Techniques Used: Immunostaining, Immunofluorescence, Staining

    Kv3.4 channel biophysical properties from naive, laminectomy and SCI. A , B , Normalized G p - V relation and steady-state inactivation relation for naive (black) and 1 week after laminectomy (blue) and SCI (red). displays mean ± SEM of best-fit
    Figure Legend Snippet: Kv3.4 channel biophysical properties from naive, laminectomy and SCI. A , B , Normalized G p - V relation and steady-state inactivation relation for naive (black) and 1 week after laminectomy (blue) and SCI (red). displays mean ± SEM of best-fit

    Techniques Used:

    Kv3.4 currents in small-diameter DRG neurons undergo dynamic changes after laminectomy and SCI
    Figure Legend Snippet: Kv3.4 currents in small-diameter DRG neurons undergo dynamic changes after laminectomy and SCI

    Techniques Used:

    2) Product Images from "K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones"

    Article Title: K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1076-16.2017

    A schematic summary of Kv3.4 function in the growth cone. A , In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca 2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K + outward currents reduce membrane excitability. B , After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca 2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca 2+ influx does not require the release of intracellular Ca 2+ from the ER (endoplasmic reticulum). C , The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca 2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca 2+ -dependent action potentials to generate Ca 2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca 2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca 2+ ] i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP 3 R, inositol 1,4,5-triphosphate receptor.
    Figure Legend Snippet: A schematic summary of Kv3.4 function in the growth cone. A , In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca 2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K + outward currents reduce membrane excitability. B , After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca 2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca 2+ influx does not require the release of intracellular Ca 2+ from the ER (endoplasmic reticulum). C , The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca 2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca 2+ -dependent action potentials to generate Ca 2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca 2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca 2+ ] i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP 3 R, inositol 1,4,5-triphosphate receptor.

    Techniques Used: Activity Assay, Binding Assay, Droplet Countercurrent Chromatography, Inhibition, Activation Assay, Concentration Assay

    Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo . A–E , The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B ), EYFP/LacZshRNA ( C ), EYFP/Kv3.4shRNA ( D ), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) ( E ). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP + commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E , Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D , Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F , Summary of projection errors of spinal commissural axons. G , The percentage of EYFP + axons with projection errors. H , The width of the ventral funiculus. I , Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M , In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA ( K ), EYFP/Kv3.4shRNA ( L ), or EYFP/Kv3.4shRNA/resKv3.4 ( M ). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero . EYFP + callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N , Measurement of axon projection to the contralateral side. Relative intensity in each region ( J , −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G , H , N , Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. * p
    Figure Legend Snippet: Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo . A–E , The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B ), EYFP/LacZshRNA ( C ), EYFP/Kv3.4shRNA ( D ), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) ( E ). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP + commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E , Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D , Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F , Summary of projection errors of spinal commissural axons. G , The percentage of EYFP + axons with projection errors. H , The width of the ventral funiculus. I , Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M , In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA ( K ), EYFP/Kv3.4shRNA ( L ), or EYFP/Kv3.4shRNA/resKv3.4 ( M ). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero . EYFP + callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N , Measurement of axon projection to the contralateral side. Relative intensity in each region ( J , −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G , H , N , Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. * p

    Techniques Used: In Vivo, Construct, Western Blot, Transfection, In Utero, Expressing, Fluorescence

    Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D , The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A ), LacZshRNA/EYFP (LacZshRNA, B ), Kv3.4shRNA/EYFP (Kv3.4shRNA, C ), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D ). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D , A′–D′ , Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A , B , In the control or LacZshRNA + neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C , Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D , Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E , After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP + neurons by that of EYFP − neurons. F , The percentage of protrusion-bearing neurons. G , The percentage of axon-bearing neurons. H , The average of axon length. I , The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP + neuron counts pooled from three independent experiments. Data are mean ± SEM. * p
    Figure Legend Snippet: Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D , The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A ), LacZshRNA/EYFP (LacZshRNA, B ), Kv3.4shRNA/EYFP (Kv3.4shRNA, C ), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D ). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D , A′–D′ , Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A , B , In the control or LacZshRNA + neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C , Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D , Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E , After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP + neurons by that of EYFP − neurons. F , The percentage of protrusion-bearing neurons. G , The percentage of axon-bearing neurons. H , The average of axon length. I , The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP + neuron counts pooled from three independent experiments. Data are mean ± SEM. * p

    Techniques Used: Construct, Cell Culture, Immunolabeling, Labeling, Expressing, Cotransfection, Fluorescence, Transfection

    Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A , B , Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) ( A ) and the bifurcation zone (BZ) of DRG neuron afferents ( B ). C , D , Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E , F , RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 ( E′ ) or TAG-1 ( F′ ). Kv3.4-IR is evident in axonal growth cones ( C″–F″ , arrows). Scale bars: A , 38 μm; B , 25 μm; C , 16 μm; D , 13 μm; E , F , 16 μm.
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A , B , Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) ( A ) and the bifurcation zone (BZ) of DRG neuron afferents ( B ). C , D , Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E , F , RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 ( E′ ) or TAG-1 ( F′ ). Kv3.4-IR is evident in axonal growth cones ( C″–F″ , arrows). Scale bars: A , 38 μm; B , 25 μm; C , 16 μm; D , 13 μm; E , F , 16 μm.

    Techniques Used: Cell Culture

    Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F , Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A , Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons ( B–F , arrowheads) is evident during HH19-HH25 but disappears at HH27. D , Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L , Transverse sections of the spinal cord at HH23 were immunostained as indicated. G , Absence of Kv1.5-IR. H , Kv4.2-IR in the somata and dendrites of motoneurons (MN). I , Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons ( J , arrow) but weak in precrossing commissural axons ( J , arrowhead). K , Absence of Kv3.2-IR. L , Kv3.3 in motoneurons. M–M″ , Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″ , Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″ , Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone ( O–O″ ) and Kv3.1b in the soma/axon shaft ( P–P″ ) of cultured dorsal spinal neurons. Q–Q″ , Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions ( Q″ ). R , Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM ( n = 8 neurons, pooled from three independent experiments done on different days). *** p
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F , Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A , Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons ( B–F , arrowheads) is evident during HH19-HH25 but disappears at HH27. D , Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L , Transverse sections of the spinal cord at HH23 were immunostained as indicated. G , Absence of Kv1.5-IR. H , Kv4.2-IR in the somata and dendrites of motoneurons (MN). I , Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons ( J , arrow) but weak in precrossing commissural axons ( J , arrowhead). K , Absence of Kv3.2-IR. L , Kv3.3 in motoneurons. M–M″ , Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″ , Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″ , Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone ( O–O″ ) and Kv3.1b in the soma/axon shaft ( P–P″ ) of cultured dorsal spinal neurons. Q–Q″ , Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions ( Q″ ). R , Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM ( n = 8 neurons, pooled from three independent experiments done on different days). *** p

    Techniques Used: Double Staining, Cell Culture, Isolation, Fluorescence

    3) Product Images from "β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses"

    Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2643-17.2018

    BACE1 and Kv3.4 colocalize in the MF pathway of the hippocampus. A , Immunofluorescence staining showing prominent Kv3.4 signal in the hilar region and in the MF tract. Staining was performed with the rabbit-anti-Kv3.4 antibody and DAPI in 1-month-old BACE1 WT (left, n = 5) and KO (right, n = 4) mice. Scale bar, 500 μm. B , Double staining for Kv3.4 (left, mouse-anti-Kv3.4 antibody) and BACE1 (middle, rabbit-anti-BACE1 antibody; Abcam) with superposition of images (right) shows a distinct colocalization in the hippocampus of 1-month-old BACE1 WT mice. Scale bar, 500 μm. Higher-magnification images below show the hilar region (right) and the end of the MF tract (left), n = 3. Scale bar, 50 μm. C , Double staining of Kv3.4 and BACE1 with higher-magnification images as described in B for hippocampal slices of age-matched KO mice, n = 3. Scale bars as in B .
    Figure Legend Snippet: BACE1 and Kv3.4 colocalize in the MF pathway of the hippocampus. A , Immunofluorescence staining showing prominent Kv3.4 signal in the hilar region and in the MF tract. Staining was performed with the rabbit-anti-Kv3.4 antibody and DAPI in 1-month-old BACE1 WT (left, n = 5) and KO (right, n = 4) mice. Scale bar, 500 μm. B , Double staining for Kv3.4 (left, mouse-anti-Kv3.4 antibody) and BACE1 (middle, rabbit-anti-BACE1 antibody; Abcam) with superposition of images (right) shows a distinct colocalization in the hippocampus of 1-month-old BACE1 WT mice. Scale bar, 500 μm. Higher-magnification images below show the hilar region (right) and the end of the MF tract (left), n = 3. Scale bar, 50 μm. C , Double staining of Kv3.4 and BACE1 with higher-magnification images as described in B for hippocampal slices of age-matched KO mice, n = 3. Scale bars as in B .

    Techniques Used: Immunofluorescence, Staining, Mouse Assay, Double Staining

    BACE1-null mice show reduced Kv3.4 level in hippocampal synapses. A , Representative Western blot of hippocampal fractions showing total, cytosolic, and synaptic fraction of 1-month-old BACE1 WT and KO mice. B , Synaptic level of the indicated proteins was quantified and normalized to corresponding β-actin levels. WT (white columns) was set to 1 for illustration. The red column shows the KO result for Kv3.4, the gray columns for other synaptic K + channels and synaptic markers. n = 3 for each genotype, * p
    Figure Legend Snippet: BACE1-null mice show reduced Kv3.4 level in hippocampal synapses. A , Representative Western blot of hippocampal fractions showing total, cytosolic, and synaptic fraction of 1-month-old BACE1 WT and KO mice. B , Synaptic level of the indicated proteins was quantified and normalized to corresponding β-actin levels. WT (white columns) was set to 1 for illustration. The red column shows the KO result for Kv3.4, the gray columns for other synaptic K + channels and synaptic markers. n = 3 for each genotype, * p

    Techniques Used: Mouse Assay, Western Blot

    Mice treated with the BACE inhibitor NB-360 show no significant decrease in Kv3.4 levels at hippocampal synapses. A , Representative Western blot of hippocampal fractions showing cytosolic and synaptic proteins of 2-month-old C57BL/6 mice, which were fed food pellets containing BACE inhibitor NB-360 or control pellets for 4 weeks. B , Synaptic level of the indicated proteins quantified and normalized to the corresponding β-actin levels. Untreated controls (white columns) were set to 1 for illustration. Red column shows Kv3.4 results from treated mice. Gray columns depict results for the BACE1 substrates CNTN2 and APP, synaptic marker proteins synapsin-1 and PSD-95, and for BACE1 protein in NB-360-fed mice. C , Scatter plot demonstrating the synaptic protein level of Kv3.4 for each investigated animal. Bar indicates mean. D , Correlation analysis of BACE1 versus Kv3.4 expression. Pearson's r = 0.79. n = 8 for treatment and control group, ** p
    Figure Legend Snippet: Mice treated with the BACE inhibitor NB-360 show no significant decrease in Kv3.4 levels at hippocampal synapses. A , Representative Western blot of hippocampal fractions showing cytosolic and synaptic proteins of 2-month-old C57BL/6 mice, which were fed food pellets containing BACE inhibitor NB-360 or control pellets for 4 weeks. B , Synaptic level of the indicated proteins quantified and normalized to the corresponding β-actin levels. Untreated controls (white columns) were set to 1 for illustration. Red column shows Kv3.4 results from treated mice. Gray columns depict results for the BACE1 substrates CNTN2 and APP, synaptic marker proteins synapsin-1 and PSD-95, and for BACE1 protein in NB-360-fed mice. C , Scatter plot demonstrating the synaptic protein level of Kv3.4 for each investigated animal. Bar indicates mean. D , Correlation analysis of BACE1 versus Kv3.4 expression. Pearson's r = 0.79. n = 8 for treatment and control group, ** p

    Techniques Used: Mouse Assay, Western Blot, Marker, Expressing

    BACE1 alters Kv3.4 trafficking in cultured hippocampal neurons. A , Left, Immunofluorescence double staining in a hippocampal neuron obtained from WT transfected with Kv3.4-EGFP and BACE1 (left: green, mouse-anti-Kv3.4 antibody; red, rabbit-anti-BACE1 antibody; Abcam; and DAPI). Scale bar, 20 μm. Right, Higher-magnification images of the indicated region along the axon. Scale bar, 10 μm. WT, n = 8 axons; KO, n = 3 axons. Double staining was performed in transfected hippocampal neurons at 6–7 DIV in three independent WT and two KO cultures. B – D , Axonal transport of Kv3.4-EGFP in transfected hippocampal neurons of WT was imaged at 6–8 DIV. Images were sampled every 530 ms for 6 min before and after bleaching the axonal segment. B , This frame of an image series shows an axon (top) and a representative part of the corresponding kymographs before (middle) and after bleaching (lower). C , D , Graphs demonstrating the cumulative probabilities for velocities of Kv3.4-EGFP vesicles moving in anterograde or retrograde direction depending on BACE1 coexpression (anterograde: Kv3.4-EGFP, n = 863, + BACE1, n = 788; bleached: Kv3.4-EGFP, n = 680, + BACE1, n = 565; retrograde: Kv3.4-EGFP, n = 875, + BACE1, n = 810; bleached: n = 378, + BACE1, n = 346). Data were obtained from three independent cultures. *** p
    Figure Legend Snippet: BACE1 alters Kv3.4 trafficking in cultured hippocampal neurons. A , Left, Immunofluorescence double staining in a hippocampal neuron obtained from WT transfected with Kv3.4-EGFP and BACE1 (left: green, mouse-anti-Kv3.4 antibody; red, rabbit-anti-BACE1 antibody; Abcam; and DAPI). Scale bar, 20 μm. Right, Higher-magnification images of the indicated region along the axon. Scale bar, 10 μm. WT, n = 8 axons; KO, n = 3 axons. Double staining was performed in transfected hippocampal neurons at 6–7 DIV in three independent WT and two KO cultures. B – D , Axonal transport of Kv3.4-EGFP in transfected hippocampal neurons of WT was imaged at 6–8 DIV. Images were sampled every 530 ms for 6 min before and after bleaching the axonal segment. B , This frame of an image series shows an axon (top) and a representative part of the corresponding kymographs before (middle) and after bleaching (lower). C , D , Graphs demonstrating the cumulative probabilities for velocities of Kv3.4-EGFP vesicles moving in anterograde or retrograde direction depending on BACE1 coexpression (anterograde: Kv3.4-EGFP, n = 863, + BACE1, n = 788; bleached: Kv3.4-EGFP, n = 680, + BACE1, n = 565; retrograde: Kv3.4-EGFP, n = 875, + BACE1, n = 810; bleached: n = 378, + BACE1, n = 346). Data were obtained from three independent cultures. *** p

    Techniques Used: Cell Culture, Immunofluorescence, Double Staining, Transfection, Mass Spectrometry

    Kv3.4 surface levels are reduced in the hippocampus of BACE1 KO mice. A , Representative Western blot of a hippocampal slice biotinylation for a WT/KO set. B , Total protein levels were densitometrically quantified, normalized to β-actin, and each KO sample was normalized to the corresponding WT that had been set to 1. Red column shows total Kv3.4 protein in KO and results for the positive control CNTN2 are illustrated in the gray column. C , Surface proteins were quantified and normalized to the corresponding levels of pan-cadherin or Na + -K + -ATPase and KO samples were normalized to WT according to B . Results of KO mice reveal a significant decrease in surface level for Kv3.4 (red columns) and a significant increase for CNTN2 (gray columns). WT/KO, n = 4 pairs, * p
    Figure Legend Snippet: Kv3.4 surface levels are reduced in the hippocampus of BACE1 KO mice. A , Representative Western blot of a hippocampal slice biotinylation for a WT/KO set. B , Total protein levels were densitometrically quantified, normalized to β-actin, and each KO sample was normalized to the corresponding WT that had been set to 1. Red column shows total Kv3.4 protein in KO and results for the positive control CNTN2 are illustrated in the gray column. C , Surface proteins were quantified and normalized to the corresponding levels of pan-cadherin or Na + -K + -ATPase and KO samples were normalized to WT according to B . Results of KO mice reveal a significant decrease in surface level for Kv3.4 (red columns) and a significant increase for CNTN2 (gray columns). WT/KO, n = 4 pairs, * p

    Techniques Used: Mouse Assay, Western Blot, Positive Control

    BACE1 amplifies Kv3.4 current and alters channel kinetics in a heterologous expression system. A1 and A2 show representative currents of a cell expressing Kv3.4 ( A1 ) or Kv3.4 with BACE1 ( A2 ) recorded with the activation protocol shown in the inset. B1 and B2 display representative currents of a Kv3.4-expressing ( B1 ) or Kv3.4 + BACE1-expressing ( B2 ) cell recorded with the inactivation protocol shown in the inset. C , The I–V relationship was generated from Kv3.4 peak currents plotted as function of test potentials from −50 mV to +30 mV, Kv3.4, n = 60; + BACE1, n = 44. D , Relative noninactivating current was calculated as steady-state current divided by peak current. Peak current was determined from the test pulse at +30 mV after a prepulse at −120 mV ( B1 , B2 , prepulse duration 500 ms). Steady-state current was obtained from recordings with a prepulse at +20 mV that activated and inactivated approximately all channels, resulting in no peak and only noninactivating steady-state current at +30 mV. This steady-state current was averaged over the last 28 ms of the test pulse (see arrow in B1 and B2 ). Kv3.4, n = 49; + BACE1, n = 36. E , Voltage-dependent activation (squares) and inactivation (triangles) curves. Activation: conductance was generated from mean peak current ( C ). The graphs present the mean conductance fitted with a Boltzmann equation and normalized to the upper asymptote. Kv3.4, n = 60; + BACE1, n = 44. Inactivation: current amplitudes of individual recordings from test pulses after prepulses of −60 mV to +20 mV recorded with the inactivation protocol ( B1 ) were fitted with a Boltzmann equation. Peak current values were normalized to the upper asymptote. Kv3.4, n = 49; + BACE1, n = 36. F , Activation and inactivation time constants were estimated from recordings with the activation protocol ( A1 ). Rise and decay at 0 mV and +10 mV were fitted using a biexponential function to determine activation and inactivation kinetics, respectively. 0 mV: Kv3.4, n = 50; + BACE1, n = 38; +10 mV: Kv3.4, n = 50; + BACE1, n = 39. Statistical significance of time constants was tested on logarithmically transformed data. For illustration, time constant means ± SEM were back-transformed to a linear scale. G , Time-dependent recovery from channel inactivation recorded with the protocol shown in the inset with varying interpulse intervals Δ t = 2 i [ms], i = 1–12. Graph shows time-dependent recovery of peak current after inactivation ( I Δt) normalized to the peak before channel inactivation ( I ). Kv3.4, n = 44; + BACE1, n = 23. H , Representative currents of a cell expressing Kv3.4 or Kv3.4 + BACE1 in response to 10 command protocols of the AP waveform with 1 Hz. I , Graphs generated from Kv3.4 peak currents recorded with the command protocol shown in H . Kv3.4, n = 50; + BACE1, n = 42. D , E Inactivation, F , G , I , Cells with peak currents
    Figure Legend Snippet: BACE1 amplifies Kv3.4 current and alters channel kinetics in a heterologous expression system. A1 and A2 show representative currents of a cell expressing Kv3.4 ( A1 ) or Kv3.4 with BACE1 ( A2 ) recorded with the activation protocol shown in the inset. B1 and B2 display representative currents of a Kv3.4-expressing ( B1 ) or Kv3.4 + BACE1-expressing ( B2 ) cell recorded with the inactivation protocol shown in the inset. C , The I–V relationship was generated from Kv3.4 peak currents plotted as function of test potentials from −50 mV to +30 mV, Kv3.4, n = 60; + BACE1, n = 44. D , Relative noninactivating current was calculated as steady-state current divided by peak current. Peak current was determined from the test pulse at +30 mV after a prepulse at −120 mV ( B1 , B2 , prepulse duration 500 ms). Steady-state current was obtained from recordings with a prepulse at +20 mV that activated and inactivated approximately all channels, resulting in no peak and only noninactivating steady-state current at +30 mV. This steady-state current was averaged over the last 28 ms of the test pulse (see arrow in B1 and B2 ). Kv3.4, n = 49; + BACE1, n = 36. E , Voltage-dependent activation (squares) and inactivation (triangles) curves. Activation: conductance was generated from mean peak current ( C ). The graphs present the mean conductance fitted with a Boltzmann equation and normalized to the upper asymptote. Kv3.4, n = 60; + BACE1, n = 44. Inactivation: current amplitudes of individual recordings from test pulses after prepulses of −60 mV to +20 mV recorded with the inactivation protocol ( B1 ) were fitted with a Boltzmann equation. Peak current values were normalized to the upper asymptote. Kv3.4, n = 49; + BACE1, n = 36. F , Activation and inactivation time constants were estimated from recordings with the activation protocol ( A1 ). Rise and decay at 0 mV and +10 mV were fitted using a biexponential function to determine activation and inactivation kinetics, respectively. 0 mV: Kv3.4, n = 50; + BACE1, n = 38; +10 mV: Kv3.4, n = 50; + BACE1, n = 39. Statistical significance of time constants was tested on logarithmically transformed data. For illustration, time constant means ± SEM were back-transformed to a linear scale. G , Time-dependent recovery from channel inactivation recorded with the protocol shown in the inset with varying interpulse intervals Δ t = 2 i [ms], i = 1–12. Graph shows time-dependent recovery of peak current after inactivation ( I Δt) normalized to the peak before channel inactivation ( I ). Kv3.4, n = 44; + BACE1, n = 23. H , Representative currents of a cell expressing Kv3.4 or Kv3.4 + BACE1 in response to 10 command protocols of the AP waveform with 1 Hz. I , Graphs generated from Kv3.4 peak currents recorded with the command protocol shown in H . Kv3.4, n = 50; + BACE1, n = 42. D , E Inactivation, F , G , I , Cells with peak currents

    Techniques Used: Expressing, Activation Assay, Generated, Mass Spectrometry, Transformation Assay

    BACE1 interacts directly with Kv3.4. A , B , Representative Western blot of a Kv3.4-IP with BACE1 co-IP ( A , n = 3) or BACE1-IP with Kv3.4 co-IP ( B , n = 2) and the corresponding isotype controls in transfected HEK293T cells. EGFP served as transfection marker. C , FRAP experiments. Example traces of BACE1-EGFP fluorescence upon coexpression of mCherry (black) or Kv3.4 (red) with corresponding fit curves using the equation I(t) = A * e − t/ τ + I 0 are shown. D , Recovery time constants of BACE1-EGFP coexpressed with the constructs as indicated below the graph. Statistical significance of time constants was tested on logarithmically transformed data. * p
    Figure Legend Snippet: BACE1 interacts directly with Kv3.4. A , B , Representative Western blot of a Kv3.4-IP with BACE1 co-IP ( A , n = 3) or BACE1-IP with Kv3.4 co-IP ( B , n = 2) and the corresponding isotype controls in transfected HEK293T cells. EGFP served as transfection marker. C , FRAP experiments. Example traces of BACE1-EGFP fluorescence upon coexpression of mCherry (black) or Kv3.4 (red) with corresponding fit curves using the equation I(t) = A * e − t/ τ + I 0 are shown. D , Recovery time constants of BACE1-EGFP coexpressed with the constructs as indicated below the graph. Statistical significance of time constants was tested on logarithmically transformed data. * p

    Techniques Used: Western Blot, Co-Immunoprecipitation Assay, Transfection, Marker, Fluorescence, Construct, Transformation Assay

    4) Product Images from "Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury"

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Quantification of Kv3.4 plasma membrane immunostaining from 2 weeks after laminectomy and SCI. A , Representative immunofluorescence sections stained with anti-Kv3.4 and pan-cadherin antibodies from laminectomy ( N = 3) and SCI ( N = 3) animals. B , Histogram
    Figure Legend Snippet: Quantification of Kv3.4 plasma membrane immunostaining from 2 weeks after laminectomy and SCI. A , Representative immunofluorescence sections stained with anti-Kv3.4 and pan-cadherin antibodies from laminectomy ( N = 3) and SCI ( N = 3) animals. B , Histogram

    Techniques Used: Immunostaining, Immunofluorescence, Staining

    Kv3.4 current expression and inactivation from naive, laminectomy, and SCI. A , Scatter plot of I p for all data on a logarithmic scale. Dashed gray line is 1 SD from the center of the naive distribution (16.1 pA). B , I p / I 500 ratio for all groups examined.
    Figure Legend Snippet: Kv3.4 current expression and inactivation from naive, laminectomy, and SCI. A , Scatter plot of I p for all data on a logarithmic scale. Dashed gray line is 1 SD from the center of the naive distribution (16.1 pA). B , I p / I 500 ratio for all groups examined.

    Techniques Used: Expressing

    Kv3.4 current phenotypes after laminectomy and SCI. A , B , Representative Kv3.4 currents from laminectomy (Lam) and SCI neurons at 1 week and 2 weeks after surgery. Pulse protocol and scale bars are as in A . C , Pie charts showing the percentages
    Figure Legend Snippet: Kv3.4 current phenotypes after laminectomy and SCI. A , B , Representative Kv3.4 currents from laminectomy (Lam) and SCI neurons at 1 week and 2 weeks after surgery. Pulse protocol and scale bars are as in A . C , Pie charts showing the percentages

    Techniques Used: Laser Capture Microdissection

    Kv3.4 channel expression and current phenotypes in DRG neurons. A , DRG section from a naive animal immunostained as indicated. B , Distribution of peak currents from naive neurons and best fit Gaussian distribution (solid line; inset: x c = 33.8 pA, w =
    Figure Legend Snippet: Kv3.4 channel expression and current phenotypes in DRG neurons. A , DRG section from a naive animal immunostained as indicated. B , Distribution of peak currents from naive neurons and best fit Gaussian distribution (solid line; inset: x c = 33.8 pA, w =

    Techniques Used: Expressing

    Dynamic clamping of a spiking DRG neuron in the SCI group. A , Cell-attached Kv3.4 currents evoked by 150 pulses to +100 mV (black traces) and the mean current (red trace). Pulse protocol details in legend. Dashed line represents 0 pA. B , Variance
    Figure Legend Snippet: Dynamic clamping of a spiking DRG neuron in the SCI group. A , Cell-attached Kv3.4 currents evoked by 150 pulses to +100 mV (black traces) and the mean current (red trace). Pulse protocol details in legend. Dashed line represents 0 pA. B , Variance

    Techniques Used:

    Early changes: laminectomy and SCI alter the inactivation properties and expression of Kv3.4 channels in DRG neurons
    Figure Legend Snippet: Early changes: laminectomy and SCI alter the inactivation properties and expression of Kv3.4 channels in DRG neurons

    Techniques Used: Expressing

    Expression of Kv3.4 mRNA and protein from naive and 2 weeks after laminectomy and SCI. A–C , Box plots of single-cell mRNA copies of Kv3.4 mRNA (black = naive; blue = laminectomy; red = SCI). Box is 25–75 percentiles with median marked
    Figure Legend Snippet: Expression of Kv3.4 mRNA and protein from naive and 2 weeks after laminectomy and SCI. A–C , Box plots of single-cell mRNA copies of Kv3.4 mRNA (black = naive; blue = laminectomy; red = SCI). Box is 25–75 percentiles with median marked

    Techniques Used: Expressing

    Kv3.4 DRG immunostaining from naive and 2 weeks laminectomy and SCI. A – C , Representative immunofluorescence sections stained with anti-Kv3.4 antibody from naive, laminectomy and SCI animals. White arrows show neurons with significant staining
    Figure Legend Snippet: Kv3.4 DRG immunostaining from naive and 2 weeks laminectomy and SCI. A – C , Representative immunofluorescence sections stained with anti-Kv3.4 antibody from naive, laminectomy and SCI animals. White arrows show neurons with significant staining

    Techniques Used: Immunostaining, Immunofluorescence, Staining

    Kv3.4 channel biophysical properties from naive, laminectomy and SCI. A , B , Normalized G p - V relation and steady-state inactivation relation for naive (black) and 1 week after laminectomy (blue) and SCI (red). displays mean ± SEM of best-fit
    Figure Legend Snippet: Kv3.4 channel biophysical properties from naive, laminectomy and SCI. A , B , Normalized G p - V relation and steady-state inactivation relation for naive (black) and 1 week after laminectomy (blue) and SCI (red). displays mean ± SEM of best-fit

    Techniques Used:

    Kv3.4 currents in small-diameter DRG neurons undergo dynamic changes after laminectomy and SCI
    Figure Legend Snippet: Kv3.4 currents in small-diameter DRG neurons undergo dynamic changes after laminectomy and SCI

    Techniques Used:

    5) Product Images from "β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses"

    Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2643-17.2018

    BACE1 and Kv3.4 colocalize in the MF pathway of the hippocampus. A , Immunofluorescence staining showing prominent Kv3.4 signal in the hilar region and in the MF tract. Staining was performed with the rabbit-anti-Kv3.4 antibody and DAPI in 1-month-old BACE1 WT (left, n = 5) and KO (right, n = 4) mice. Scale bar, 500 μm. B , Double staining for Kv3.4 (left, mouse-anti-Kv3.4 antibody) and BACE1 (middle, rabbit-anti-BACE1 antibody; Abcam) with superposition of images (right) shows a distinct colocalization in the hippocampus of 1-month-old BACE1 WT mice. Scale bar, 500 μm. Higher-magnification images below show the hilar region (right) and the end of the MF tract (left), n = 3. Scale bar, 50 μm. C , Double staining of Kv3.4 and BACE1 with higher-magnification images as described in B for hippocampal slices of age-matched KO mice, n = 3. Scale bars as in B .
    Figure Legend Snippet: BACE1 and Kv3.4 colocalize in the MF pathway of the hippocampus. A , Immunofluorescence staining showing prominent Kv3.4 signal in the hilar region and in the MF tract. Staining was performed with the rabbit-anti-Kv3.4 antibody and DAPI in 1-month-old BACE1 WT (left, n = 5) and KO (right, n = 4) mice. Scale bar, 500 μm. B , Double staining for Kv3.4 (left, mouse-anti-Kv3.4 antibody) and BACE1 (middle, rabbit-anti-BACE1 antibody; Abcam) with superposition of images (right) shows a distinct colocalization in the hippocampus of 1-month-old BACE1 WT mice. Scale bar, 500 μm. Higher-magnification images below show the hilar region (right) and the end of the MF tract (left), n = 3. Scale bar, 50 μm. C , Double staining of Kv3.4 and BACE1 with higher-magnification images as described in B for hippocampal slices of age-matched KO mice, n = 3. Scale bars as in B .

    Techniques Used: Immunofluorescence, Staining, Mouse Assay, Double Staining

    BACE1-null mice show reduced Kv3.4 level in hippocampal synapses. A , Representative Western blot of hippocampal fractions showing total, cytosolic, and synaptic fraction of 1-month-old BACE1 WT and KO mice. B , Synaptic level of the indicated proteins was quantified and normalized to corresponding β-actin levels. WT (white columns) was set to 1 for illustration. The red column shows the KO result for Kv3.4, the gray columns for other synaptic K + channels and synaptic markers. n = 3 for each genotype, * p
    Figure Legend Snippet: BACE1-null mice show reduced Kv3.4 level in hippocampal synapses. A , Representative Western blot of hippocampal fractions showing total, cytosolic, and synaptic fraction of 1-month-old BACE1 WT and KO mice. B , Synaptic level of the indicated proteins was quantified and normalized to corresponding β-actin levels. WT (white columns) was set to 1 for illustration. The red column shows the KO result for Kv3.4, the gray columns for other synaptic K + channels and synaptic markers. n = 3 for each genotype, * p

    Techniques Used: Mouse Assay, Western Blot

    Mice treated with the BACE inhibitor NB-360 show no significant decrease in Kv3.4 levels at hippocampal synapses. A , Representative Western blot of hippocampal fractions showing cytosolic and synaptic proteins of 2-month-old C57BL/6 mice, which were fed food pellets containing BACE inhibitor NB-360 or control pellets for 4 weeks. B , Synaptic level of the indicated proteins quantified and normalized to the corresponding β-actin levels. Untreated controls (white columns) were set to 1 for illustration. Red column shows Kv3.4 results from treated mice. Gray columns depict results for the BACE1 substrates CNTN2 and APP, synaptic marker proteins synapsin-1 and PSD-95, and for BACE1 protein in NB-360-fed mice. C , Scatter plot demonstrating the synaptic protein level of Kv3.4 for each investigated animal. Bar indicates mean. D , Correlation analysis of BACE1 versus Kv3.4 expression. Pearson's r = 0.79. n = 8 for treatment and control group, ** p
    Figure Legend Snippet: Mice treated with the BACE inhibitor NB-360 show no significant decrease in Kv3.4 levels at hippocampal synapses. A , Representative Western blot of hippocampal fractions showing cytosolic and synaptic proteins of 2-month-old C57BL/6 mice, which were fed food pellets containing BACE inhibitor NB-360 or control pellets for 4 weeks. B , Synaptic level of the indicated proteins quantified and normalized to the corresponding β-actin levels. Untreated controls (white columns) were set to 1 for illustration. Red column shows Kv3.4 results from treated mice. Gray columns depict results for the BACE1 substrates CNTN2 and APP, synaptic marker proteins synapsin-1 and PSD-95, and for BACE1 protein in NB-360-fed mice. C , Scatter plot demonstrating the synaptic protein level of Kv3.4 for each investigated animal. Bar indicates mean. D , Correlation analysis of BACE1 versus Kv3.4 expression. Pearson's r = 0.79. n = 8 for treatment and control group, ** p

    Techniques Used: Mouse Assay, Western Blot, Marker, Expressing

    BACE1 alters Kv3.4 trafficking in cultured hippocampal neurons. A , Left, Immunofluorescence double staining in a hippocampal neuron obtained from WT transfected with Kv3.4-EGFP and BACE1 (left: green, mouse-anti-Kv3.4 antibody; red, rabbit-anti-BACE1 antibody; Abcam; and DAPI). Scale bar, 20 μm. Right, Higher-magnification images of the indicated region along the axon. Scale bar, 10 μm. WT, n = 8 axons; KO, n = 3 axons. Double staining was performed in transfected hippocampal neurons at 6–7 DIV in three independent WT and two KO cultures. B – D , Axonal transport of Kv3.4-EGFP in transfected hippocampal neurons of WT was imaged at 6–8 DIV. Images were sampled every 530 ms for 6 min before and after bleaching the axonal segment. B , This frame of an image series shows an axon (top) and a representative part of the corresponding kymographs before (middle) and after bleaching (lower). C , D , Graphs demonstrating the cumulative probabilities for velocities of Kv3.4-EGFP vesicles moving in anterograde or retrograde direction depending on BACE1 coexpression (anterograde: Kv3.4-EGFP, n = 863, + BACE1, n = 788; bleached: Kv3.4-EGFP, n = 680, + BACE1, n = 565; retrograde: Kv3.4-EGFP, n = 875, + BACE1, n = 810; bleached: n = 378, + BACE1, n = 346). Data were obtained from three independent cultures. *** p
    Figure Legend Snippet: BACE1 alters Kv3.4 trafficking in cultured hippocampal neurons. A , Left, Immunofluorescence double staining in a hippocampal neuron obtained from WT transfected with Kv3.4-EGFP and BACE1 (left: green, mouse-anti-Kv3.4 antibody; red, rabbit-anti-BACE1 antibody; Abcam; and DAPI). Scale bar, 20 μm. Right, Higher-magnification images of the indicated region along the axon. Scale bar, 10 μm. WT, n = 8 axons; KO, n = 3 axons. Double staining was performed in transfected hippocampal neurons at 6–7 DIV in three independent WT and two KO cultures. B – D , Axonal transport of Kv3.4-EGFP in transfected hippocampal neurons of WT was imaged at 6–8 DIV. Images were sampled every 530 ms for 6 min before and after bleaching the axonal segment. B , This frame of an image series shows an axon (top) and a representative part of the corresponding kymographs before (middle) and after bleaching (lower). C , D , Graphs demonstrating the cumulative probabilities for velocities of Kv3.4-EGFP vesicles moving in anterograde or retrograde direction depending on BACE1 coexpression (anterograde: Kv3.4-EGFP, n = 863, + BACE1, n = 788; bleached: Kv3.4-EGFP, n = 680, + BACE1, n = 565; retrograde: Kv3.4-EGFP, n = 875, + BACE1, n = 810; bleached: n = 378, + BACE1, n = 346). Data were obtained from three independent cultures. *** p

    Techniques Used: Cell Culture, Immunofluorescence, Double Staining, Transfection

    Kv3.4 surface levels are reduced in the hippocampus of BACE1 KO mice. A , Representative Western blot of a hippocampal slice biotinylation for a WT/KO set. B , Total protein levels were densitometrically quantified, normalized to β-actin, and each KO sample was normalized to the corresponding WT that had been set to 1. Red column shows total Kv3.4 protein in KO and results for the positive control CNTN2 are illustrated in the gray column. C , Surface proteins were quantified and normalized to the corresponding levels of pan-cadherin or Na + -K + -ATPase and KO samples were normalized to WT according to B . Results of KO mice reveal a significant decrease in surface level for Kv3.4 (red columns) and a significant increase for CNTN2 (gray columns). WT/KO, n = 4 pairs, * p
    Figure Legend Snippet: Kv3.4 surface levels are reduced in the hippocampus of BACE1 KO mice. A , Representative Western blot of a hippocampal slice biotinylation for a WT/KO set. B , Total protein levels were densitometrically quantified, normalized to β-actin, and each KO sample was normalized to the corresponding WT that had been set to 1. Red column shows total Kv3.4 protein in KO and results for the positive control CNTN2 are illustrated in the gray column. C , Surface proteins were quantified and normalized to the corresponding levels of pan-cadherin or Na + -K + -ATPase and KO samples were normalized to WT according to B . Results of KO mice reveal a significant decrease in surface level for Kv3.4 (red columns) and a significant increase for CNTN2 (gray columns). WT/KO, n = 4 pairs, * p

    Techniques Used: Mouse Assay, Western Blot, Positive Control

    BACE1 amplifies Kv3.4 current and alters channel kinetics in a heterologous expression system. A1 and A2 show representative currents of a cell expressing Kv3.4 ( A1 ) or Kv3.4 with BACE1 ( A2 ) recorded with the activation protocol shown in the inset. B1 and B2 display representative currents of a Kv3.4-expressing ( B1 ) or Kv3.4 + BACE1-expressing ( B2 ) cell recorded with the inactivation protocol shown in the inset. C , The I–V relationship was generated from Kv3.4 peak currents plotted as function of test potentials from −50 mV to +30 mV, Kv3.4, n = 60; + BACE1, n = 44. D , Relative noninactivating current was calculated as steady-state current divided by peak current. Peak current was determined from the test pulse at +30 mV after a prepulse at −120 mV ( B1 , B2 , prepulse duration 500 ms). Steady-state current was obtained from recordings with a prepulse at +20 mV that activated and inactivated approximately all channels, resulting in no peak and only noninactivating steady-state current at +30 mV. This steady-state current was averaged over the last 28 ms of the test pulse (see arrow in B1 and B2 ). Kv3.4, n = 49; + BACE1, n = 36. E , Voltage-dependent activation (squares) and inactivation (triangles) curves. Activation: conductance was generated from mean peak current ( C ). The graphs present the mean conductance fitted with a Boltzmann equation and normalized to the upper asymptote. Kv3.4, n = 60; + BACE1, n = 44. Inactivation: current amplitudes of individual recordings from test pulses after prepulses of −60 mV to +20 mV recorded with the inactivation protocol ( B1 ) were fitted with a Boltzmann equation. Peak current values were normalized to the upper asymptote. Kv3.4, n = 49; + BACE1, n = 36. F , Activation and inactivation time constants were estimated from recordings with the activation protocol ( A1 ). Rise and decay at 0 mV and +10 mV were fitted using a biexponential function to determine activation and inactivation kinetics, respectively. 0 mV: Kv3.4, n = 50; + BACE1, n = 38; +10 mV: Kv3.4, n = 50; + BACE1, n = 39. Statistical significance of time constants was tested on logarithmically transformed data. For illustration, time constant means ± SEM were back-transformed to a linear scale. G , Time-dependent recovery from channel inactivation recorded with the protocol shown in the inset with varying interpulse intervals Δ t = 2 i [ms], i = 1–12. Graph shows time-dependent recovery of peak current after inactivation ( I Δt) normalized to the peak before channel inactivation ( I ). Kv3.4, n = 44; + BACE1, n = 23. H , Representative currents of a cell expressing Kv3.4 or Kv3.4 + BACE1 in response to 10 command protocols of the AP waveform with 1 Hz. I , Graphs generated from Kv3.4 peak currents recorded with the command protocol shown in H . Kv3.4, n = 50; + BACE1, n = 42. D , E Inactivation, F , G , I , Cells with peak currents
    Figure Legend Snippet: BACE1 amplifies Kv3.4 current and alters channel kinetics in a heterologous expression system. A1 and A2 show representative currents of a cell expressing Kv3.4 ( A1 ) or Kv3.4 with BACE1 ( A2 ) recorded with the activation protocol shown in the inset. B1 and B2 display representative currents of a Kv3.4-expressing ( B1 ) or Kv3.4 + BACE1-expressing ( B2 ) cell recorded with the inactivation protocol shown in the inset. C , The I–V relationship was generated from Kv3.4 peak currents plotted as function of test potentials from −50 mV to +30 mV, Kv3.4, n = 60; + BACE1, n = 44. D , Relative noninactivating current was calculated as steady-state current divided by peak current. Peak current was determined from the test pulse at +30 mV after a prepulse at −120 mV ( B1 , B2 , prepulse duration 500 ms). Steady-state current was obtained from recordings with a prepulse at +20 mV that activated and inactivated approximately all channels, resulting in no peak and only noninactivating steady-state current at +30 mV. This steady-state current was averaged over the last 28 ms of the test pulse (see arrow in B1 and B2 ). Kv3.4, n = 49; + BACE1, n = 36. E , Voltage-dependent activation (squares) and inactivation (triangles) curves. Activation: conductance was generated from mean peak current ( C ). The graphs present the mean conductance fitted with a Boltzmann equation and normalized to the upper asymptote. Kv3.4, n = 60; + BACE1, n = 44. Inactivation: current amplitudes of individual recordings from test pulses after prepulses of −60 mV to +20 mV recorded with the inactivation protocol ( B1 ) were fitted with a Boltzmann equation. Peak current values were normalized to the upper asymptote. Kv3.4, n = 49; + BACE1, n = 36. F , Activation and inactivation time constants were estimated from recordings with the activation protocol ( A1 ). Rise and decay at 0 mV and +10 mV were fitted using a biexponential function to determine activation and inactivation kinetics, respectively. 0 mV: Kv3.4, n = 50; + BACE1, n = 38; +10 mV: Kv3.4, n = 50; + BACE1, n = 39. Statistical significance of time constants was tested on logarithmically transformed data. For illustration, time constant means ± SEM were back-transformed to a linear scale. G , Time-dependent recovery from channel inactivation recorded with the protocol shown in the inset with varying interpulse intervals Δ t = 2 i [ms], i = 1–12. Graph shows time-dependent recovery of peak current after inactivation ( I Δt) normalized to the peak before channel inactivation ( I ). Kv3.4, n = 44; + BACE1, n = 23. H , Representative currents of a cell expressing Kv3.4 or Kv3.4 + BACE1 in response to 10 command protocols of the AP waveform with 1 Hz. I , Graphs generated from Kv3.4 peak currents recorded with the command protocol shown in H . Kv3.4, n = 50; + BACE1, n = 42. D , E Inactivation, F , G , I , Cells with peak currents

    Techniques Used: Expressing, Activation Assay, Generated, Transformation Assay

    BACE1 interacts directly with Kv3.4. A , B , Representative Western blot of a Kv3.4-IP with BACE1 co-IP ( A , n = 3) or BACE1-IP with Kv3.4 co-IP ( B , n = 2) and the corresponding isotype controls in transfected HEK293T cells. EGFP served as transfection marker. C , FRAP experiments. Example traces of BACE1-EGFP fluorescence upon coexpression of mCherry (black) or Kv3.4 (red) with corresponding fit curves using the equation I(t) = A * e − t/ τ + I 0 are shown. D , Recovery time constants of BACE1-EGFP coexpressed with the constructs as indicated below the graph. Statistical significance of time constants was tested on logarithmically transformed data. * p
    Figure Legend Snippet: BACE1 interacts directly with Kv3.4. A , B , Representative Western blot of a Kv3.4-IP with BACE1 co-IP ( A , n = 3) or BACE1-IP with Kv3.4 co-IP ( B , n = 2) and the corresponding isotype controls in transfected HEK293T cells. EGFP served as transfection marker. C , FRAP experiments. Example traces of BACE1-EGFP fluorescence upon coexpression of mCherry (black) or Kv3.4 (red) with corresponding fit curves using the equation I(t) = A * e − t/ τ + I 0 are shown. D , Recovery time constants of BACE1-EGFP coexpressed with the constructs as indicated below the graph. Statistical significance of time constants was tested on logarithmically transformed data. * p

    Techniques Used: Western Blot, Co-Immunoprecipitation Assay, Transfection, Marker, Fluorescence, Construct, Transformation Assay

    6) Product Images from "Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury"

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Quantification of Kv3.4 plasma membrane immunostaining from 2 weeks after laminectomy and SCI. A , Representative immunofluorescence sections stained with anti-Kv3.4 and pan-cadherin antibodies from laminectomy ( N = 3) and SCI ( N = 3) animals. B , Histogram
    Figure Legend Snippet: Quantification of Kv3.4 plasma membrane immunostaining from 2 weeks after laminectomy and SCI. A , Representative immunofluorescence sections stained with anti-Kv3.4 and pan-cadherin antibodies from laminectomy ( N = 3) and SCI ( N = 3) animals. B , Histogram

    Techniques Used: Immunostaining, Immunofluorescence, Staining

    Kv3.4 current expression and inactivation from naive, laminectomy, and SCI. A , Scatter plot of I p for all data on a logarithmic scale. Dashed gray line is 1 SD from the center of the naive distribution (16.1 pA). B , I p / I 500 ratio for all groups examined.
    Figure Legend Snippet: Kv3.4 current expression and inactivation from naive, laminectomy, and SCI. A , Scatter plot of I p for all data on a logarithmic scale. Dashed gray line is 1 SD from the center of the naive distribution (16.1 pA). B , I p / I 500 ratio for all groups examined.

    Techniques Used: Expressing

    Kv3.4 current phenotypes after laminectomy and SCI. A , B , Representative Kv3.4 currents from laminectomy (Lam) and SCI neurons at 1 week and 2 weeks after surgery. Pulse protocol and scale bars are as in A . C , Pie charts showing the percentages
    Figure Legend Snippet: Kv3.4 current phenotypes after laminectomy and SCI. A , B , Representative Kv3.4 currents from laminectomy (Lam) and SCI neurons at 1 week and 2 weeks after surgery. Pulse protocol and scale bars are as in A . C , Pie charts showing the percentages

    Techniques Used: Laser Capture Microdissection

    Kv3.4 channel expression and current phenotypes in DRG neurons. A , DRG section from a naive animal immunostained as indicated. B , Distribution of peak currents from naive neurons and best fit Gaussian distribution (solid line; inset: x c = 33.8 pA, w =
    Figure Legend Snippet: Kv3.4 channel expression and current phenotypes in DRG neurons. A , DRG section from a naive animal immunostained as indicated. B , Distribution of peak currents from naive neurons and best fit Gaussian distribution (solid line; inset: x c = 33.8 pA, w =

    Techniques Used: Expressing

    Dynamic clamping of a spiking DRG neuron in the SCI group. A , Cell-attached Kv3.4 currents evoked by 150 pulses to +100 mV (black traces) and the mean current (red trace). Pulse protocol details in legend. Dashed line represents 0 pA. B , Variance
    Figure Legend Snippet: Dynamic clamping of a spiking DRG neuron in the SCI group. A , Cell-attached Kv3.4 currents evoked by 150 pulses to +100 mV (black traces) and the mean current (red trace). Pulse protocol details in legend. Dashed line represents 0 pA. B , Variance

    Techniques Used:

    Early changes: laminectomy and SCI alter the inactivation properties and expression of Kv3.4 channels in DRG neurons
    Figure Legend Snippet: Early changes: laminectomy and SCI alter the inactivation properties and expression of Kv3.4 channels in DRG neurons

    Techniques Used: Expressing

    Expression of Kv3.4 mRNA and protein from naive and 2 weeks after laminectomy and SCI. A–C , Box plots of single-cell mRNA copies of Kv3.4 mRNA (black = naive; blue = laminectomy; red = SCI). Box is 25–75 percentiles with median marked
    Figure Legend Snippet: Expression of Kv3.4 mRNA and protein from naive and 2 weeks after laminectomy and SCI. A–C , Box plots of single-cell mRNA copies of Kv3.4 mRNA (black = naive; blue = laminectomy; red = SCI). Box is 25–75 percentiles with median marked

    Techniques Used: Expressing

    Kv3.4 DRG immunostaining from naive and 2 weeks laminectomy and SCI. A – C , Representative immunofluorescence sections stained with anti-Kv3.4 antibody from naive, laminectomy and SCI animals. White arrows show neurons with significant staining
    Figure Legend Snippet: Kv3.4 DRG immunostaining from naive and 2 weeks laminectomy and SCI. A – C , Representative immunofluorescence sections stained with anti-Kv3.4 antibody from naive, laminectomy and SCI animals. White arrows show neurons with significant staining

    Techniques Used: Immunostaining, Immunofluorescence, Staining

    Kv3.4 channel biophysical properties from naive, laminectomy and SCI. A , B , Normalized G p - V relation and steady-state inactivation relation for naive (black) and 1 week after laminectomy (blue) and SCI (red). displays mean ± SEM of best-fit
    Figure Legend Snippet: Kv3.4 channel biophysical properties from naive, laminectomy and SCI. A , B , Normalized G p - V relation and steady-state inactivation relation for naive (black) and 1 week after laminectomy (blue) and SCI (red). displays mean ± SEM of best-fit

    Techniques Used:

    Kv3.4 currents in small-diameter DRG neurons undergo dynamic changes after laminectomy and SCI
    Figure Legend Snippet: Kv3.4 currents in small-diameter DRG neurons undergo dynamic changes after laminectomy and SCI

    Techniques Used:

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    Alomone Labs goat anti kv3 4 antibody
    Quantification of <t>Kv3.4</t> plasma membrane immunostaining from 2 weeks after laminectomy and SCI. A , Representative immunofluorescence sections stained with anti-Kv3.4 and pan-cadherin antibodies from laminectomy ( N = 3) and SCI ( N = 3) animals. B , Histogram
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    Quantification of Kv3.4 plasma membrane immunostaining from 2 weeks after laminectomy and SCI. A , Representative immunofluorescence sections stained with anti-Kv3.4 and pan-cadherin antibodies from laminectomy ( N = 3) and SCI ( N = 3) animals. B , Histogram

    Journal: The Journal of Neuroscience

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Figure Lengend Snippet: Quantification of Kv3.4 plasma membrane immunostaining from 2 weeks after laminectomy and SCI. A , Representative immunofluorescence sections stained with anti-Kv3.4 and pan-cadherin antibodies from laminectomy ( N = 3) and SCI ( N = 3) animals. B , Histogram

    Article Snippet: Sections were then incubated with primary goat anti-Kv3.4 antibody (1:1000, Alomone Labs Catalog #APC-019 RRID:AB_2040172) overnight at 4°C.

    Techniques: Immunostaining, Immunofluorescence, Staining

    Kv3.4 current expression and inactivation from naive, laminectomy, and SCI. A , Scatter plot of I p for all data on a logarithmic scale. Dashed gray line is 1 SD from the center of the naive distribution (16.1 pA). B , I p / I 500 ratio for all groups examined.

    Journal: The Journal of Neuroscience

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Figure Lengend Snippet: Kv3.4 current expression and inactivation from naive, laminectomy, and SCI. A , Scatter plot of I p for all data on a logarithmic scale. Dashed gray line is 1 SD from the center of the naive distribution (16.1 pA). B , I p / I 500 ratio for all groups examined.

    Article Snippet: Sections were then incubated with primary goat anti-Kv3.4 antibody (1:1000, Alomone Labs Catalog #APC-019 RRID:AB_2040172) overnight at 4°C.

    Techniques: Expressing

    Kv3.4 current phenotypes after laminectomy and SCI. A , B , Representative Kv3.4 currents from laminectomy (Lam) and SCI neurons at 1 week and 2 weeks after surgery. Pulse protocol and scale bars are as in A . C , Pie charts showing the percentages

    Journal: The Journal of Neuroscience

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Figure Lengend Snippet: Kv3.4 current phenotypes after laminectomy and SCI. A , B , Representative Kv3.4 currents from laminectomy (Lam) and SCI neurons at 1 week and 2 weeks after surgery. Pulse protocol and scale bars are as in A . C , Pie charts showing the percentages

    Article Snippet: Sections were then incubated with primary goat anti-Kv3.4 antibody (1:1000, Alomone Labs Catalog #APC-019 RRID:AB_2040172) overnight at 4°C.

    Techniques: Laser Capture Microdissection

    Kv3.4 channel expression and current phenotypes in DRG neurons. A , DRG section from a naive animal immunostained as indicated. B , Distribution of peak currents from naive neurons and best fit Gaussian distribution (solid line; inset: x c = 33.8 pA, w =

    Journal: The Journal of Neuroscience

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Figure Lengend Snippet: Kv3.4 channel expression and current phenotypes in DRG neurons. A , DRG section from a naive animal immunostained as indicated. B , Distribution of peak currents from naive neurons and best fit Gaussian distribution (solid line; inset: x c = 33.8 pA, w =

    Article Snippet: Sections were then incubated with primary goat anti-Kv3.4 antibody (1:1000, Alomone Labs Catalog #APC-019 RRID:AB_2040172) overnight at 4°C.

    Techniques: Expressing

    Dynamic clamping of a spiking DRG neuron in the SCI group. A , Cell-attached Kv3.4 currents evoked by 150 pulses to +100 mV (black traces) and the mean current (red trace). Pulse protocol details in legend. Dashed line represents 0 pA. B , Variance

    Journal: The Journal of Neuroscience

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Figure Lengend Snippet: Dynamic clamping of a spiking DRG neuron in the SCI group. A , Cell-attached Kv3.4 currents evoked by 150 pulses to +100 mV (black traces) and the mean current (red trace). Pulse protocol details in legend. Dashed line represents 0 pA. B , Variance

    Article Snippet: Sections were then incubated with primary goat anti-Kv3.4 antibody (1:1000, Alomone Labs Catalog #APC-019 RRID:AB_2040172) overnight at 4°C.

    Techniques:

    Early changes: laminectomy and SCI alter the inactivation properties and expression of Kv3.4 channels in DRG neurons

    Journal: The Journal of Neuroscience

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Figure Lengend Snippet: Early changes: laminectomy and SCI alter the inactivation properties and expression of Kv3.4 channels in DRG neurons

    Article Snippet: Sections were then incubated with primary goat anti-Kv3.4 antibody (1:1000, Alomone Labs Catalog #APC-019 RRID:AB_2040172) overnight at 4°C.

    Techniques: Expressing

    Expression of Kv3.4 mRNA and protein from naive and 2 weeks after laminectomy and SCI. A–C , Box plots of single-cell mRNA copies of Kv3.4 mRNA (black = naive; blue = laminectomy; red = SCI). Box is 25–75 percentiles with median marked

    Journal: The Journal of Neuroscience

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Figure Lengend Snippet: Expression of Kv3.4 mRNA and protein from naive and 2 weeks after laminectomy and SCI. A–C , Box plots of single-cell mRNA copies of Kv3.4 mRNA (black = naive; blue = laminectomy; red = SCI). Box is 25–75 percentiles with median marked

    Article Snippet: Sections were then incubated with primary goat anti-Kv3.4 antibody (1:1000, Alomone Labs Catalog #APC-019 RRID:AB_2040172) overnight at 4°C.

    Techniques: Expressing

    Kv3.4 DRG immunostaining from naive and 2 weeks laminectomy and SCI. A – C , Representative immunofluorescence sections stained with anti-Kv3.4 antibody from naive, laminectomy and SCI animals. White arrows show neurons with significant staining

    Journal: The Journal of Neuroscience

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Figure Lengend Snippet: Kv3.4 DRG immunostaining from naive and 2 weeks laminectomy and SCI. A – C , Representative immunofluorescence sections stained with anti-Kv3.4 antibody from naive, laminectomy and SCI animals. White arrows show neurons with significant staining

    Article Snippet: Sections were then incubated with primary goat anti-Kv3.4 antibody (1:1000, Alomone Labs Catalog #APC-019 RRID:AB_2040172) overnight at 4°C.

    Techniques: Immunostaining, Immunofluorescence, Staining

    Kv3.4 channel biophysical properties from naive, laminectomy and SCI. A , B , Normalized G p - V relation and steady-state inactivation relation for naive (black) and 1 week after laminectomy (blue) and SCI (red). displays mean ± SEM of best-fit

    Journal: The Journal of Neuroscience

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Figure Lengend Snippet: Kv3.4 channel biophysical properties from naive, laminectomy and SCI. A , B , Normalized G p - V relation and steady-state inactivation relation for naive (black) and 1 week after laminectomy (blue) and SCI (red). displays mean ± SEM of best-fit

    Article Snippet: Sections were then incubated with primary goat anti-Kv3.4 antibody (1:1000, Alomone Labs Catalog #APC-019 RRID:AB_2040172) overnight at 4°C.

    Techniques:

    Kv3.4 currents in small-diameter DRG neurons undergo dynamic changes after laminectomy and SCI

    Journal: The Journal of Neuroscience

    Article Title: Dysregulation of Kv3.4 Channels in Dorsal Root Ganglia Following Spinal Cord Injury

    doi: 10.1523/JNEUROSCI.1594-14.2015

    Figure Lengend Snippet: Kv3.4 currents in small-diameter DRG neurons undergo dynamic changes after laminectomy and SCI

    Article Snippet: Sections were then incubated with primary goat anti-Kv3.4 antibody (1:1000, Alomone Labs Catalog #APC-019 RRID:AB_2040172) overnight at 4°C.

    Techniques:

    A schematic summary of Kv3.4 function in the growth cone. A , In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca 2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K + outward currents reduce membrane excitability. B , After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca 2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca 2+ influx does not require the release of intracellular Ca 2+ from the ER (endoplasmic reticulum). C , The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca 2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca 2+ -dependent action potentials to generate Ca 2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca 2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca 2+ ] i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP 3 R, inositol 1,4,5-triphosphate receptor.

    Journal: The Journal of Neuroscience

    Article Title: K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: A schematic summary of Kv3.4 function in the growth cone. A , In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca 2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K + outward currents reduce membrane excitability. B , After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca 2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca 2+ influx does not require the release of intracellular Ca 2+ from the ER (endoplasmic reticulum). C , The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca 2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca 2+ -dependent action potentials to generate Ca 2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca 2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca 2+ ] i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP 3 R, inositol 1,4,5-triphosphate receptor.

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( ).

    Techniques: Activity Assay, Binding Assay, Droplet Countercurrent Chromatography, Inhibition, Activation Assay, Concentration Assay

    Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo . A–E , The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B ), EYFP/LacZshRNA ( C ), EYFP/Kv3.4shRNA ( D ), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) ( E ). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP + commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E , Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D , Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F , Summary of projection errors of spinal commissural axons. G , The percentage of EYFP + axons with projection errors. H , The width of the ventral funiculus. I , Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M , In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA ( K ), EYFP/Kv3.4shRNA ( L ), or EYFP/Kv3.4shRNA/resKv3.4 ( M ). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero . EYFP + callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N , Measurement of axon projection to the contralateral side. Relative intensity in each region ( J , −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G , H , N , Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. * p

    Journal: The Journal of Neuroscience

    Article Title: K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo . A–E , The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B ), EYFP/LacZshRNA ( C ), EYFP/Kv3.4shRNA ( D ), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) ( E ). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP + commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E , Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D , Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F , Summary of projection errors of spinal commissural axons. G , The percentage of EYFP + axons with projection errors. H , The width of the ventral funiculus. I , Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M , In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA ( K ), EYFP/Kv3.4shRNA ( L ), or EYFP/Kv3.4shRNA/resKv3.4 ( M ). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero . EYFP + callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N , Measurement of axon projection to the contralateral side. Relative intensity in each region ( J , −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G , H , N , Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. * p

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( ).

    Techniques: In Vivo, Construct, Western Blot, Transfection, In Utero, Expressing, Fluorescence

    Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D , The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A ), LacZshRNA/EYFP (LacZshRNA, B ), Kv3.4shRNA/EYFP (Kv3.4shRNA, C ), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D ). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D , A′–D′ , Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A , B , In the control or LacZshRNA + neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C , Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D , Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E , After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP + neurons by that of EYFP − neurons. F , The percentage of protrusion-bearing neurons. G , The percentage of axon-bearing neurons. H , The average of axon length. I , The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP + neuron counts pooled from three independent experiments. Data are mean ± SEM. * p

    Journal: The Journal of Neuroscience

    Article Title: K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D , The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A ), LacZshRNA/EYFP (LacZshRNA, B ), Kv3.4shRNA/EYFP (Kv3.4shRNA, C ), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D ). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D , A′–D′ , Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A , B , In the control or LacZshRNA + neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C , Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D , Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E , After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP + neurons by that of EYFP − neurons. F , The percentage of protrusion-bearing neurons. G , The percentage of axon-bearing neurons. H , The average of axon length. I , The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP + neuron counts pooled from three independent experiments. Data are mean ± SEM. * p

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( ).

    Techniques: Construct, Cell Culture, Immunolabeling, Labeling, Expressing, Cotransfection, Fluorescence, Transfection

    Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A , B , Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) ( A ) and the bifurcation zone (BZ) of DRG neuron afferents ( B ). C , D , Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E , F , RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 ( E′ ) or TAG-1 ( F′ ). Kv3.4-IR is evident in axonal growth cones ( C″–F″ , arrows). Scale bars: A , 38 μm; B , 25 μm; C , 16 μm; D , 13 μm; E , F , 16 μm.

    Journal: The Journal of Neuroscience

    Article Title: K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A , B , Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) ( A ) and the bifurcation zone (BZ) of DRG neuron afferents ( B ). C , D , Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E , F , RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 ( E′ ) or TAG-1 ( F′ ). Kv3.4-IR is evident in axonal growth cones ( C″–F″ , arrows). Scale bars: A , 38 μm; B , 25 μm; C , 16 μm; D , 13 μm; E , F , 16 μm.

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( ).

    Techniques: Cell Culture

    Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F , Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A , Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons ( B–F , arrowheads) is evident during HH19-HH25 but disappears at HH27. D , Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L , Transverse sections of the spinal cord at HH23 were immunostained as indicated. G , Absence of Kv1.5-IR. H , Kv4.2-IR in the somata and dendrites of motoneurons (MN). I , Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons ( J , arrow) but weak in precrossing commissural axons ( J , arrowhead). K , Absence of Kv3.2-IR. L , Kv3.3 in motoneurons. M–M″ , Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″ , Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″ , Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone ( O–O″ ) and Kv3.1b in the soma/axon shaft ( P–P″ ) of cultured dorsal spinal neurons. Q–Q″ , Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions ( Q″ ). R , Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM ( n = 8 neurons, pooled from three independent experiments done on different days). *** p

    Journal: The Journal of Neuroscience

    Article Title: K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F , Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A , Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons ( B–F , arrowheads) is evident during HH19-HH25 but disappears at HH27. D , Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L , Transverse sections of the spinal cord at HH23 were immunostained as indicated. G , Absence of Kv1.5-IR. H , Kv4.2-IR in the somata and dendrites of motoneurons (MN). I , Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons ( J , arrow) but weak in precrossing commissural axons ( J , arrowhead). K , Absence of Kv3.2-IR. L , Kv3.3 in motoneurons. M–M″ , Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″ , Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″ , Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone ( O–O″ ) and Kv3.1b in the soma/axon shaft ( P–P″ ) of cultured dorsal spinal neurons. Q–Q″ , Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions ( Q″ ). R , Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM ( n = 8 neurons, pooled from three independent experiments done on different days). *** p

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( ).

    Techniques: Double Staining, Cell Culture, Isolation, Fluorescence

    BACE1 and Kv3.4 colocalize in the MF pathway of the hippocampus. A , Immunofluorescence staining showing prominent Kv3.4 signal in the hilar region and in the MF tract. Staining was performed with the rabbit-anti-Kv3.4 antibody and DAPI in 1-month-old BACE1 WT (left, n = 5) and KO (right, n = 4) mice. Scale bar, 500 μm. B , Double staining for Kv3.4 (left, mouse-anti-Kv3.4 antibody) and BACE1 (middle, rabbit-anti-BACE1 antibody; Abcam) with superposition of images (right) shows a distinct colocalization in the hippocampus of 1-month-old BACE1 WT mice. Scale bar, 500 μm. Higher-magnification images below show the hilar region (right) and the end of the MF tract (left), n = 3. Scale bar, 50 μm. C , Double staining of Kv3.4 and BACE1 with higher-magnification images as described in B for hippocampal slices of age-matched KO mice, n = 3. Scale bars as in B .

    Journal: The Journal of Neuroscience

    Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

    doi: 10.1523/JNEUROSCI.2643-17.2018

    Figure Lengend Snippet: BACE1 and Kv3.4 colocalize in the MF pathway of the hippocampus. A , Immunofluorescence staining showing prominent Kv3.4 signal in the hilar region and in the MF tract. Staining was performed with the rabbit-anti-Kv3.4 antibody and DAPI in 1-month-old BACE1 WT (left, n = 5) and KO (right, n = 4) mice. Scale bar, 500 μm. B , Double staining for Kv3.4 (left, mouse-anti-Kv3.4 antibody) and BACE1 (middle, rabbit-anti-BACE1 antibody; Abcam) with superposition of images (right) shows a distinct colocalization in the hippocampus of 1-month-old BACE1 WT mice. Scale bar, 500 μm. Higher-magnification images below show the hilar region (right) and the end of the MF tract (left), n = 3. Scale bar, 50 μm. C , Double staining of Kv3.4 and BACE1 with higher-magnification images as described in B for hippocampal slices of age-matched KO mice, n = 3. Scale bars as in B .

    Article Snippet: The following antibodies were used in this work: rabbit-anti-Kv3.4 (APC-019; Alomone Laboratories), mouse-anti-Kv3.4 (75-112; NeuroMab), rabbit-anti-BACE1 (ab108394; Abcam), goat-anti-BACE1 (ab11028; Abcam), rabbit-anti-BACE (D10E5, 5606; Cell Signaling Technology), rabbit-anti-pan-cadherin (4068; Cell Signaling Technology), rabbit-anti-Na+ -K+ -ATPase α1 (3010; Cell Signaling Technology), rabbit-anti-V5 (ab9116; Abcam), goat-anti-HA (ab9134; Abcam), mouse-anti-β-actin-HRP (A3854; Sigma-Aldrich), goat-anti-CNTN2 (AF4439; R & D Systems), mouse-anti-KCa 1.1 (ab192759; Abcam), rabbit-anti-KCa 2.3 (APC-025; Alomone Laboratories), rabbit-anti-synapsin-1 XP (D12G5, 5297; Cell Signaling Technology), mouse-anti-PSD-95 (75-028; NeuroMab), mouse-anti-NR2B (73-097/73-101; NeuroMab), rabbit-anti-APP (C66; ), goat-anti-rabbit-HRP (ab6721; Abcam), rabbit-anti-goat-HRP (ab6741; Abcam), rabbit-anti-mouse-HRP (ab6728; Abcam), donkey-anti-rabbit-HRP (NA934; GE Healthcare), sheep-anti-mouse-HRP (NA931; GE Healthcare), donkey-anti-goat-HRP (705-035-147; Jackson Laboratories), mouse-anti-rabbit-HRP (18-8816-31, TrueBlot; Rockland), IRDye 680RD goat-anti-mouse IgG (P/N 926-68070; LI-COR), IRDye 800CW donkey-anti-rabbit IgG (P/N 926-32213; LI-COR), goat-anti-mouse-Alexa Fluor 488 (A-11029; Invitrogen), and goat-anti-rabbit-Cy3 (111-165-144; Dianova).

    Techniques: Immunofluorescence, Staining, Mouse Assay, Double Staining

    BACE1-null mice show reduced Kv3.4 level in hippocampal synapses. A , Representative Western blot of hippocampal fractions showing total, cytosolic, and synaptic fraction of 1-month-old BACE1 WT and KO mice. B , Synaptic level of the indicated proteins was quantified and normalized to corresponding β-actin levels. WT (white columns) was set to 1 for illustration. The red column shows the KO result for Kv3.4, the gray columns for other synaptic K + channels and synaptic markers. n = 3 for each genotype, * p

    Journal: The Journal of Neuroscience

    Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

    doi: 10.1523/JNEUROSCI.2643-17.2018

    Figure Lengend Snippet: BACE1-null mice show reduced Kv3.4 level in hippocampal synapses. A , Representative Western blot of hippocampal fractions showing total, cytosolic, and synaptic fraction of 1-month-old BACE1 WT and KO mice. B , Synaptic level of the indicated proteins was quantified and normalized to corresponding β-actin levels. WT (white columns) was set to 1 for illustration. The red column shows the KO result for Kv3.4, the gray columns for other synaptic K + channels and synaptic markers. n = 3 for each genotype, * p

    Article Snippet: The following antibodies were used in this work: rabbit-anti-Kv3.4 (APC-019; Alomone Laboratories), mouse-anti-Kv3.4 (75-112; NeuroMab), rabbit-anti-BACE1 (ab108394; Abcam), goat-anti-BACE1 (ab11028; Abcam), rabbit-anti-BACE (D10E5, 5606; Cell Signaling Technology), rabbit-anti-pan-cadherin (4068; Cell Signaling Technology), rabbit-anti-Na+ -K+ -ATPase α1 (3010; Cell Signaling Technology), rabbit-anti-V5 (ab9116; Abcam), goat-anti-HA (ab9134; Abcam), mouse-anti-β-actin-HRP (A3854; Sigma-Aldrich), goat-anti-CNTN2 (AF4439; R & D Systems), mouse-anti-KCa 1.1 (ab192759; Abcam), rabbit-anti-KCa 2.3 (APC-025; Alomone Laboratories), rabbit-anti-synapsin-1 XP (D12G5, 5297; Cell Signaling Technology), mouse-anti-PSD-95 (75-028; NeuroMab), mouse-anti-NR2B (73-097/73-101; NeuroMab), rabbit-anti-APP (C66; ), goat-anti-rabbit-HRP (ab6721; Abcam), rabbit-anti-goat-HRP (ab6741; Abcam), rabbit-anti-mouse-HRP (ab6728; Abcam), donkey-anti-rabbit-HRP (NA934; GE Healthcare), sheep-anti-mouse-HRP (NA931; GE Healthcare), donkey-anti-goat-HRP (705-035-147; Jackson Laboratories), mouse-anti-rabbit-HRP (18-8816-31, TrueBlot; Rockland), IRDye 680RD goat-anti-mouse IgG (P/N 926-68070; LI-COR), IRDye 800CW donkey-anti-rabbit IgG (P/N 926-32213; LI-COR), goat-anti-mouse-Alexa Fluor 488 (A-11029; Invitrogen), and goat-anti-rabbit-Cy3 (111-165-144; Dianova).

    Techniques: Mouse Assay, Western Blot

    Mice treated with the BACE inhibitor NB-360 show no significant decrease in Kv3.4 levels at hippocampal synapses. A , Representative Western blot of hippocampal fractions showing cytosolic and synaptic proteins of 2-month-old C57BL/6 mice, which were fed food pellets containing BACE inhibitor NB-360 or control pellets for 4 weeks. B , Synaptic level of the indicated proteins quantified and normalized to the corresponding β-actin levels. Untreated controls (white columns) were set to 1 for illustration. Red column shows Kv3.4 results from treated mice. Gray columns depict results for the BACE1 substrates CNTN2 and APP, synaptic marker proteins synapsin-1 and PSD-95, and for BACE1 protein in NB-360-fed mice. C , Scatter plot demonstrating the synaptic protein level of Kv3.4 for each investigated animal. Bar indicates mean. D , Correlation analysis of BACE1 versus Kv3.4 expression. Pearson's r = 0.79. n = 8 for treatment and control group, ** p

    Journal: The Journal of Neuroscience

    Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

    doi: 10.1523/JNEUROSCI.2643-17.2018

    Figure Lengend Snippet: Mice treated with the BACE inhibitor NB-360 show no significant decrease in Kv3.4 levels at hippocampal synapses. A , Representative Western blot of hippocampal fractions showing cytosolic and synaptic proteins of 2-month-old C57BL/6 mice, which were fed food pellets containing BACE inhibitor NB-360 or control pellets for 4 weeks. B , Synaptic level of the indicated proteins quantified and normalized to the corresponding β-actin levels. Untreated controls (white columns) were set to 1 for illustration. Red column shows Kv3.4 results from treated mice. Gray columns depict results for the BACE1 substrates CNTN2 and APP, synaptic marker proteins synapsin-1 and PSD-95, and for BACE1 protein in NB-360-fed mice. C , Scatter plot demonstrating the synaptic protein level of Kv3.4 for each investigated animal. Bar indicates mean. D , Correlation analysis of BACE1 versus Kv3.4 expression. Pearson's r = 0.79. n = 8 for treatment and control group, ** p

    Article Snippet: The following antibodies were used in this work: rabbit-anti-Kv3.4 (APC-019; Alomone Laboratories), mouse-anti-Kv3.4 (75-112; NeuroMab), rabbit-anti-BACE1 (ab108394; Abcam), goat-anti-BACE1 (ab11028; Abcam), rabbit-anti-BACE (D10E5, 5606; Cell Signaling Technology), rabbit-anti-pan-cadherin (4068; Cell Signaling Technology), rabbit-anti-Na+ -K+ -ATPase α1 (3010; Cell Signaling Technology), rabbit-anti-V5 (ab9116; Abcam), goat-anti-HA (ab9134; Abcam), mouse-anti-β-actin-HRP (A3854; Sigma-Aldrich), goat-anti-CNTN2 (AF4439; R & D Systems), mouse-anti-KCa 1.1 (ab192759; Abcam), rabbit-anti-KCa 2.3 (APC-025; Alomone Laboratories), rabbit-anti-synapsin-1 XP (D12G5, 5297; Cell Signaling Technology), mouse-anti-PSD-95 (75-028; NeuroMab), mouse-anti-NR2B (73-097/73-101; NeuroMab), rabbit-anti-APP (C66; ), goat-anti-rabbit-HRP (ab6721; Abcam), rabbit-anti-goat-HRP (ab6741; Abcam), rabbit-anti-mouse-HRP (ab6728; Abcam), donkey-anti-rabbit-HRP (NA934; GE Healthcare), sheep-anti-mouse-HRP (NA931; GE Healthcare), donkey-anti-goat-HRP (705-035-147; Jackson Laboratories), mouse-anti-rabbit-HRP (18-8816-31, TrueBlot; Rockland), IRDye 680RD goat-anti-mouse IgG (P/N 926-68070; LI-COR), IRDye 800CW donkey-anti-rabbit IgG (P/N 926-32213; LI-COR), goat-anti-mouse-Alexa Fluor 488 (A-11029; Invitrogen), and goat-anti-rabbit-Cy3 (111-165-144; Dianova).

    Techniques: Mouse Assay, Western Blot, Marker, Expressing

    BACE1 alters Kv3.4 trafficking in cultured hippocampal neurons. A , Left, Immunofluorescence double staining in a hippocampal neuron obtained from WT transfected with Kv3.4-EGFP and BACE1 (left: green, mouse-anti-Kv3.4 antibody; red, rabbit-anti-BACE1 antibody; Abcam; and DAPI). Scale bar, 20 μm. Right, Higher-magnification images of the indicated region along the axon. Scale bar, 10 μm. WT, n = 8 axons; KO, n = 3 axons. Double staining was performed in transfected hippocampal neurons at 6–7 DIV in three independent WT and two KO cultures. B – D , Axonal transport of Kv3.4-EGFP in transfected hippocampal neurons of WT was imaged at 6–8 DIV. Images were sampled every 530 ms for 6 min before and after bleaching the axonal segment. B , This frame of an image series shows an axon (top) and a representative part of the corresponding kymographs before (middle) and after bleaching (lower). C , D , Graphs demonstrating the cumulative probabilities for velocities of Kv3.4-EGFP vesicles moving in anterograde or retrograde direction depending on BACE1 coexpression (anterograde: Kv3.4-EGFP, n = 863, + BACE1, n = 788; bleached: Kv3.4-EGFP, n = 680, + BACE1, n = 565; retrograde: Kv3.4-EGFP, n = 875, + BACE1, n = 810; bleached: n = 378, + BACE1, n = 346). Data were obtained from three independent cultures. *** p

    Journal: The Journal of Neuroscience

    Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

    doi: 10.1523/JNEUROSCI.2643-17.2018

    Figure Lengend Snippet: BACE1 alters Kv3.4 trafficking in cultured hippocampal neurons. A , Left, Immunofluorescence double staining in a hippocampal neuron obtained from WT transfected with Kv3.4-EGFP and BACE1 (left: green, mouse-anti-Kv3.4 antibody; red, rabbit-anti-BACE1 antibody; Abcam; and DAPI). Scale bar, 20 μm. Right, Higher-magnification images of the indicated region along the axon. Scale bar, 10 μm. WT, n = 8 axons; KO, n = 3 axons. Double staining was performed in transfected hippocampal neurons at 6–7 DIV in three independent WT and two KO cultures. B – D , Axonal transport of Kv3.4-EGFP in transfected hippocampal neurons of WT was imaged at 6–8 DIV. Images were sampled every 530 ms for 6 min before and after bleaching the axonal segment. B , This frame of an image series shows an axon (top) and a representative part of the corresponding kymographs before (middle) and after bleaching (lower). C , D , Graphs demonstrating the cumulative probabilities for velocities of Kv3.4-EGFP vesicles moving in anterograde or retrograde direction depending on BACE1 coexpression (anterograde: Kv3.4-EGFP, n = 863, + BACE1, n = 788; bleached: Kv3.4-EGFP, n = 680, + BACE1, n = 565; retrograde: Kv3.4-EGFP, n = 875, + BACE1, n = 810; bleached: n = 378, + BACE1, n = 346). Data were obtained from three independent cultures. *** p

    Article Snippet: The following antibodies were used in this work: rabbit-anti-Kv3.4 (APC-019; Alomone Laboratories), mouse-anti-Kv3.4 (75-112; NeuroMab), rabbit-anti-BACE1 (ab108394; Abcam), goat-anti-BACE1 (ab11028; Abcam), rabbit-anti-BACE (D10E5, 5606; Cell Signaling Technology), rabbit-anti-pan-cadherin (4068; Cell Signaling Technology), rabbit-anti-Na+ -K+ -ATPase α1 (3010; Cell Signaling Technology), rabbit-anti-V5 (ab9116; Abcam), goat-anti-HA (ab9134; Abcam), mouse-anti-β-actin-HRP (A3854; Sigma-Aldrich), goat-anti-CNTN2 (AF4439; R & D Systems), mouse-anti-KCa 1.1 (ab192759; Abcam), rabbit-anti-KCa 2.3 (APC-025; Alomone Laboratories), rabbit-anti-synapsin-1 XP (D12G5, 5297; Cell Signaling Technology), mouse-anti-PSD-95 (75-028; NeuroMab), mouse-anti-NR2B (73-097/73-101; NeuroMab), rabbit-anti-APP (C66; ), goat-anti-rabbit-HRP (ab6721; Abcam), rabbit-anti-goat-HRP (ab6741; Abcam), rabbit-anti-mouse-HRP (ab6728; Abcam), donkey-anti-rabbit-HRP (NA934; GE Healthcare), sheep-anti-mouse-HRP (NA931; GE Healthcare), donkey-anti-goat-HRP (705-035-147; Jackson Laboratories), mouse-anti-rabbit-HRP (18-8816-31, TrueBlot; Rockland), IRDye 680RD goat-anti-mouse IgG (P/N 926-68070; LI-COR), IRDye 800CW donkey-anti-rabbit IgG (P/N 926-32213; LI-COR), goat-anti-mouse-Alexa Fluor 488 (A-11029; Invitrogen), and goat-anti-rabbit-Cy3 (111-165-144; Dianova).

    Techniques: Cell Culture, Immunofluorescence, Double Staining, Transfection, Mass Spectrometry

    Kv3.4 surface levels are reduced in the hippocampus of BACE1 KO mice. A , Representative Western blot of a hippocampal slice biotinylation for a WT/KO set. B , Total protein levels were densitometrically quantified, normalized to β-actin, and each KO sample was normalized to the corresponding WT that had been set to 1. Red column shows total Kv3.4 protein in KO and results for the positive control CNTN2 are illustrated in the gray column. C , Surface proteins were quantified and normalized to the corresponding levels of pan-cadherin or Na + -K + -ATPase and KO samples were normalized to WT according to B . Results of KO mice reveal a significant decrease in surface level for Kv3.4 (red columns) and a significant increase for CNTN2 (gray columns). WT/KO, n = 4 pairs, * p

    Journal: The Journal of Neuroscience

    Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

    doi: 10.1523/JNEUROSCI.2643-17.2018

    Figure Lengend Snippet: Kv3.4 surface levels are reduced in the hippocampus of BACE1 KO mice. A , Representative Western blot of a hippocampal slice biotinylation for a WT/KO set. B , Total protein levels were densitometrically quantified, normalized to β-actin, and each KO sample was normalized to the corresponding WT that had been set to 1. Red column shows total Kv3.4 protein in KO and results for the positive control CNTN2 are illustrated in the gray column. C , Surface proteins were quantified and normalized to the corresponding levels of pan-cadherin or Na + -K + -ATPase and KO samples were normalized to WT according to B . Results of KO mice reveal a significant decrease in surface level for Kv3.4 (red columns) and a significant increase for CNTN2 (gray columns). WT/KO, n = 4 pairs, * p

    Article Snippet: The following antibodies were used in this work: rabbit-anti-Kv3.4 (APC-019; Alomone Laboratories), mouse-anti-Kv3.4 (75-112; NeuroMab), rabbit-anti-BACE1 (ab108394; Abcam), goat-anti-BACE1 (ab11028; Abcam), rabbit-anti-BACE (D10E5, 5606; Cell Signaling Technology), rabbit-anti-pan-cadherin (4068; Cell Signaling Technology), rabbit-anti-Na+ -K+ -ATPase α1 (3010; Cell Signaling Technology), rabbit-anti-V5 (ab9116; Abcam), goat-anti-HA (ab9134; Abcam), mouse-anti-β-actin-HRP (A3854; Sigma-Aldrich), goat-anti-CNTN2 (AF4439; R & D Systems), mouse-anti-KCa 1.1 (ab192759; Abcam), rabbit-anti-KCa 2.3 (APC-025; Alomone Laboratories), rabbit-anti-synapsin-1 XP (D12G5, 5297; Cell Signaling Technology), mouse-anti-PSD-95 (75-028; NeuroMab), mouse-anti-NR2B (73-097/73-101; NeuroMab), rabbit-anti-APP (C66; ), goat-anti-rabbit-HRP (ab6721; Abcam), rabbit-anti-goat-HRP (ab6741; Abcam), rabbit-anti-mouse-HRP (ab6728; Abcam), donkey-anti-rabbit-HRP (NA934; GE Healthcare), sheep-anti-mouse-HRP (NA931; GE Healthcare), donkey-anti-goat-HRP (705-035-147; Jackson Laboratories), mouse-anti-rabbit-HRP (18-8816-31, TrueBlot; Rockland), IRDye 680RD goat-anti-mouse IgG (P/N 926-68070; LI-COR), IRDye 800CW donkey-anti-rabbit IgG (P/N 926-32213; LI-COR), goat-anti-mouse-Alexa Fluor 488 (A-11029; Invitrogen), and goat-anti-rabbit-Cy3 (111-165-144; Dianova).

    Techniques: Mouse Assay, Western Blot, Positive Control

    BACE1 amplifies Kv3.4 current and alters channel kinetics in a heterologous expression system. A1 and A2 show representative currents of a cell expressing Kv3.4 ( A1 ) or Kv3.4 with BACE1 ( A2 ) recorded with the activation protocol shown in the inset. B1 and B2 display representative currents of a Kv3.4-expressing ( B1 ) or Kv3.4 + BACE1-expressing ( B2 ) cell recorded with the inactivation protocol shown in the inset. C , The I–V relationship was generated from Kv3.4 peak currents plotted as function of test potentials from −50 mV to +30 mV, Kv3.4, n = 60; + BACE1, n = 44. D , Relative noninactivating current was calculated as steady-state current divided by peak current. Peak current was determined from the test pulse at +30 mV after a prepulse at −120 mV ( B1 , B2 , prepulse duration 500 ms). Steady-state current was obtained from recordings with a prepulse at +20 mV that activated and inactivated approximately all channels, resulting in no peak and only noninactivating steady-state current at +30 mV. This steady-state current was averaged over the last 28 ms of the test pulse (see arrow in B1 and B2 ). Kv3.4, n = 49; + BACE1, n = 36. E , Voltage-dependent activation (squares) and inactivation (triangles) curves. Activation: conductance was generated from mean peak current ( C ). The graphs present the mean conductance fitted with a Boltzmann equation and normalized to the upper asymptote. Kv3.4, n = 60; + BACE1, n = 44. Inactivation: current amplitudes of individual recordings from test pulses after prepulses of −60 mV to +20 mV recorded with the inactivation protocol ( B1 ) were fitted with a Boltzmann equation. Peak current values were normalized to the upper asymptote. Kv3.4, n = 49; + BACE1, n = 36. F , Activation and inactivation time constants were estimated from recordings with the activation protocol ( A1 ). Rise and decay at 0 mV and +10 mV were fitted using a biexponential function to determine activation and inactivation kinetics, respectively. 0 mV: Kv3.4, n = 50; + BACE1, n = 38; +10 mV: Kv3.4, n = 50; + BACE1, n = 39. Statistical significance of time constants was tested on logarithmically transformed data. For illustration, time constant means ± SEM were back-transformed to a linear scale. G , Time-dependent recovery from channel inactivation recorded with the protocol shown in the inset with varying interpulse intervals Δ t = 2 i [ms], i = 1–12. Graph shows time-dependent recovery of peak current after inactivation ( I Δt) normalized to the peak before channel inactivation ( I ). Kv3.4, n = 44; + BACE1, n = 23. H , Representative currents of a cell expressing Kv3.4 or Kv3.4 + BACE1 in response to 10 command protocols of the AP waveform with 1 Hz. I , Graphs generated from Kv3.4 peak currents recorded with the command protocol shown in H . Kv3.4, n = 50; + BACE1, n = 42. D , E Inactivation, F , G , I , Cells with peak currents

    Journal: The Journal of Neuroscience

    Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

    doi: 10.1523/JNEUROSCI.2643-17.2018

    Figure Lengend Snippet: BACE1 amplifies Kv3.4 current and alters channel kinetics in a heterologous expression system. A1 and A2 show representative currents of a cell expressing Kv3.4 ( A1 ) or Kv3.4 with BACE1 ( A2 ) recorded with the activation protocol shown in the inset. B1 and B2 display representative currents of a Kv3.4-expressing ( B1 ) or Kv3.4 + BACE1-expressing ( B2 ) cell recorded with the inactivation protocol shown in the inset. C , The I–V relationship was generated from Kv3.4 peak currents plotted as function of test potentials from −50 mV to +30 mV, Kv3.4, n = 60; + BACE1, n = 44. D , Relative noninactivating current was calculated as steady-state current divided by peak current. Peak current was determined from the test pulse at +30 mV after a prepulse at −120 mV ( B1 , B2 , prepulse duration 500 ms). Steady-state current was obtained from recordings with a prepulse at +20 mV that activated and inactivated approximately all channels, resulting in no peak and only noninactivating steady-state current at +30 mV. This steady-state current was averaged over the last 28 ms of the test pulse (see arrow in B1 and B2 ). Kv3.4, n = 49; + BACE1, n = 36. E , Voltage-dependent activation (squares) and inactivation (triangles) curves. Activation: conductance was generated from mean peak current ( C ). The graphs present the mean conductance fitted with a Boltzmann equation and normalized to the upper asymptote. Kv3.4, n = 60; + BACE1, n = 44. Inactivation: current amplitudes of individual recordings from test pulses after prepulses of −60 mV to +20 mV recorded with the inactivation protocol ( B1 ) were fitted with a Boltzmann equation. Peak current values were normalized to the upper asymptote. Kv3.4, n = 49; + BACE1, n = 36. F , Activation and inactivation time constants were estimated from recordings with the activation protocol ( A1 ). Rise and decay at 0 mV and +10 mV were fitted using a biexponential function to determine activation and inactivation kinetics, respectively. 0 mV: Kv3.4, n = 50; + BACE1, n = 38; +10 mV: Kv3.4, n = 50; + BACE1, n = 39. Statistical significance of time constants was tested on logarithmically transformed data. For illustration, time constant means ± SEM were back-transformed to a linear scale. G , Time-dependent recovery from channel inactivation recorded with the protocol shown in the inset with varying interpulse intervals Δ t = 2 i [ms], i = 1–12. Graph shows time-dependent recovery of peak current after inactivation ( I Δt) normalized to the peak before channel inactivation ( I ). Kv3.4, n = 44; + BACE1, n = 23. H , Representative currents of a cell expressing Kv3.4 or Kv3.4 + BACE1 in response to 10 command protocols of the AP waveform with 1 Hz. I , Graphs generated from Kv3.4 peak currents recorded with the command protocol shown in H . Kv3.4, n = 50; + BACE1, n = 42. D , E Inactivation, F , G , I , Cells with peak currents

    Article Snippet: The following antibodies were used in this work: rabbit-anti-Kv3.4 (APC-019; Alomone Laboratories), mouse-anti-Kv3.4 (75-112; NeuroMab), rabbit-anti-BACE1 (ab108394; Abcam), goat-anti-BACE1 (ab11028; Abcam), rabbit-anti-BACE (D10E5, 5606; Cell Signaling Technology), rabbit-anti-pan-cadherin (4068; Cell Signaling Technology), rabbit-anti-Na+ -K+ -ATPase α1 (3010; Cell Signaling Technology), rabbit-anti-V5 (ab9116; Abcam), goat-anti-HA (ab9134; Abcam), mouse-anti-β-actin-HRP (A3854; Sigma-Aldrich), goat-anti-CNTN2 (AF4439; R & D Systems), mouse-anti-KCa 1.1 (ab192759; Abcam), rabbit-anti-KCa 2.3 (APC-025; Alomone Laboratories), rabbit-anti-synapsin-1 XP (D12G5, 5297; Cell Signaling Technology), mouse-anti-PSD-95 (75-028; NeuroMab), mouse-anti-NR2B (73-097/73-101; NeuroMab), rabbit-anti-APP (C66; ), goat-anti-rabbit-HRP (ab6721; Abcam), rabbit-anti-goat-HRP (ab6741; Abcam), rabbit-anti-mouse-HRP (ab6728; Abcam), donkey-anti-rabbit-HRP (NA934; GE Healthcare), sheep-anti-mouse-HRP (NA931; GE Healthcare), donkey-anti-goat-HRP (705-035-147; Jackson Laboratories), mouse-anti-rabbit-HRP (18-8816-31, TrueBlot; Rockland), IRDye 680RD goat-anti-mouse IgG (P/N 926-68070; LI-COR), IRDye 800CW donkey-anti-rabbit IgG (P/N 926-32213; LI-COR), goat-anti-mouse-Alexa Fluor 488 (A-11029; Invitrogen), and goat-anti-rabbit-Cy3 (111-165-144; Dianova).

    Techniques: Expressing, Activation Assay, Generated, Mass Spectrometry, Transformation Assay

    BACE1 interacts directly with Kv3.4. A , B , Representative Western blot of a Kv3.4-IP with BACE1 co-IP ( A , n = 3) or BACE1-IP with Kv3.4 co-IP ( B , n = 2) and the corresponding isotype controls in transfected HEK293T cells. EGFP served as transfection marker. C , FRAP experiments. Example traces of BACE1-EGFP fluorescence upon coexpression of mCherry (black) or Kv3.4 (red) with corresponding fit curves using the equation I(t) = A * e − t/ τ + I 0 are shown. D , Recovery time constants of BACE1-EGFP coexpressed with the constructs as indicated below the graph. Statistical significance of time constants was tested on logarithmically transformed data. * p

    Journal: The Journal of Neuroscience

    Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses

    doi: 10.1523/JNEUROSCI.2643-17.2018

    Figure Lengend Snippet: BACE1 interacts directly with Kv3.4. A , B , Representative Western blot of a Kv3.4-IP with BACE1 co-IP ( A , n = 3) or BACE1-IP with Kv3.4 co-IP ( B , n = 2) and the corresponding isotype controls in transfected HEK293T cells. EGFP served as transfection marker. C , FRAP experiments. Example traces of BACE1-EGFP fluorescence upon coexpression of mCherry (black) or Kv3.4 (red) with corresponding fit curves using the equation I(t) = A * e − t/ τ + I 0 are shown. D , Recovery time constants of BACE1-EGFP coexpressed with the constructs as indicated below the graph. Statistical significance of time constants was tested on logarithmically transformed data. * p

    Article Snippet: The following antibodies were used in this work: rabbit-anti-Kv3.4 (APC-019; Alomone Laboratories), mouse-anti-Kv3.4 (75-112; NeuroMab), rabbit-anti-BACE1 (ab108394; Abcam), goat-anti-BACE1 (ab11028; Abcam), rabbit-anti-BACE (D10E5, 5606; Cell Signaling Technology), rabbit-anti-pan-cadherin (4068; Cell Signaling Technology), rabbit-anti-Na+ -K+ -ATPase α1 (3010; Cell Signaling Technology), rabbit-anti-V5 (ab9116; Abcam), goat-anti-HA (ab9134; Abcam), mouse-anti-β-actin-HRP (A3854; Sigma-Aldrich), goat-anti-CNTN2 (AF4439; R & D Systems), mouse-anti-KCa 1.1 (ab192759; Abcam), rabbit-anti-KCa 2.3 (APC-025; Alomone Laboratories), rabbit-anti-synapsin-1 XP (D12G5, 5297; Cell Signaling Technology), mouse-anti-PSD-95 (75-028; NeuroMab), mouse-anti-NR2B (73-097/73-101; NeuroMab), rabbit-anti-APP (C66; ), goat-anti-rabbit-HRP (ab6721; Abcam), rabbit-anti-goat-HRP (ab6741; Abcam), rabbit-anti-mouse-HRP (ab6728; Abcam), donkey-anti-rabbit-HRP (NA934; GE Healthcare), sheep-anti-mouse-HRP (NA931; GE Healthcare), donkey-anti-goat-HRP (705-035-147; Jackson Laboratories), mouse-anti-rabbit-HRP (18-8816-31, TrueBlot; Rockland), IRDye 680RD goat-anti-mouse IgG (P/N 926-68070; LI-COR), IRDye 800CW donkey-anti-rabbit IgG (P/N 926-32213; LI-COR), goat-anti-mouse-Alexa Fluor 488 (A-11029; Invitrogen), and goat-anti-rabbit-Cy3 (111-165-144; Dianova).

    Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection, Marker, Fluorescence, Construct, Transformation Assay