ip3r1  (Alomone Labs)

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

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

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

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

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:

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

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

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

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:

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:

Journal: bioRxiv

Article Title: Reducing voltage-dependent potassium channel Kv3.4 levels ameliorates synapse loss in a mouse model of Alzheimer’s disease

doi: 10.1101/2021.11.24.469829

Figure Lengend Snippet: Representative immunostaining images of K v 3.4 in neurons expressing either tdTomato or eYFP (A B). For comparison purposes, tdTomato and eYFP cell bodies are visualised in grey pseudocolour and Kv3.4 staining in magenta. Compared to the tdTomato-filled neuron, the eYFP-filled neuron shows lower K v 3.4 immunoreactivity. Analysis of K v 3.4 immunostaining intensity revealed a 21% reduction in staining intensity in eYFP-filled cells relative to tdTomato-filled cells (C), confirming the action of U6.sgRNA(mK v 3.4).CMV.saCas9 at knocking down K v 3.4 levels. * p

Article Snippet: Membranes were incubated with primary antibody (anti-KCNC4; Alomone #APC-019; 1:200) overnight at 4 °C in blocking buffer.

Techniques: Immunostaining, Expressing, Staining

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

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

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

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

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

Journal: Frontiers in Cellular Neuroscience

Article Title: Differential Hippocampal Expression of BDNF Isoforms and Their Receptors Under Diverse Configurations of the Serotonergic System in a Mice Model of Increased Neuronal Survival

doi: 10.3389/fncel.2019.00384

Figure Lengend Snippet: Effect of 8-OH-DPAT on the protein expression of molecules from the BDNF signaling pathway in the HC, determined by Western Blot. (A) mBDNF. (B) TrkB. (C) proBDNF. (D) p75 in hyposerotonergic (PCPA-treated) or control (vehicle-treated) mice receiving the 5-HT 1 A agonist 8-OH-DPAT (striped bars) or NaCl (white bars). (E) Representative membrane showing signal for p75 (75 kDa) and tubuline (52 kDa). 1: Control-NaCl. 2: Control-DPAT. 3: PCPA-NaCl. 4: PCPA-DPAT. Data are expressed as mean ± S.E.M., n = 6 per experimental group. ∗ p

Article Snippet: Western Blotting Membranes were incubated for 1 h with blocking solution (5% milk in TBST) and then probed overnight at 4°C with mouse anti-BDNF (1:2000; Icosagen; 327-100 clone 3C11), rabbit anti-p75 (1:700; Alomone Labs; ANT-007), rabbit anti-TrkB (1:700; Alomone Labs; ANT-019), and rabbit anti-proBDNF (1:250; Abcam; ab72440) in TBST. β-III Tubulin was used as a loading control (1:2500; R & D Systems).

Techniques: Expressing, Western Blot, Mouse Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Differential Hippocampal Expression of BDNF Isoforms and Their Receptors Under Diverse Configurations of the Serotonergic System in a Mice Model of Increased Neuronal Survival

doi: 10.3389/fncel.2019.00384

Figure Lengend Snippet: Protein expression of molecules from the BDNF signaling pathway determined by Western Blot and representative membranes. (A) mBDNF. (B) TrkB. (C) proBDNF. (D) p75 in hyposerotonergic (PCPA-treated) or control (vehicle-treated) mice for 4 weeks. (E–H) Representative membrane showing signal for TrkB, p75, pro-BDNF, and mBDNF in Control and PCPA-treatd mice. Data are expressed as mean ± S.E.M., n = 11 (control) and 12 (PCPA). ∗ p

Article Snippet: Western Blotting Membranes were incubated for 1 h with blocking solution (5% milk in TBST) and then probed overnight at 4°C with mouse anti-BDNF (1:2000; Icosagen; 327-100 clone 3C11), rabbit anti-p75 (1:700; Alomone Labs; ANT-007), rabbit anti-TrkB (1:700; Alomone Labs; ANT-019), and rabbit anti-proBDNF (1:250; Abcam; ab72440) in TBST. β-III Tubulin was used as a loading control (1:2500; R & D Systems).

Techniques: Expressing, Western Blot, Mouse Assay