rabbit anti nt3  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti nt3
    Sortilin-mediated binding and internalisation of <t>NT3</t> and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.
    Rabbit Anti Nt3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
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    85/100 stars

    Images

    1) Product Images from "Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons"

    Article Title: Proneurotrophin-3 may induce Sortilin dependent death in inner ear neurons

    Journal: The European journal of neuroscience

    doi: 10.1111/j.1460-9568.2010.07556.x

    Sortilin-mediated binding and internalisation of NT3 and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.
    Figure Legend Snippet: Sortilin-mediated binding and internalisation of NT3 and proNT3 (A–J) HEK293 cells transfected with Sortilin, p75 NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C) , proNT3 (D–F) , or GST-NT3pro (G–J) ) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A) , but internalisation was detected of both proNT3 (D) and GST-NT3pro (G) . The uptake of proNT3 could be inhibited by addition of NTS (E) . Cells transfected with p75 NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F) , but no binding of GST-NT3pro (I) . HEK293 human embryonic kidney 293 cells , NT3 neurotrophin-3 , proNT3 proform of NT3 , p75 NTR p75 neurotrophin receptor , NTS neurotensin , GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.

    Techniques Used: Binding Assay, Transfection, Incubation, Immunofluorescence

    Crosslinking and precipitation of p75 NTR :Sortilin:proNT3 complexes HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75 NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75 NTR , and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75 NTR . The expression of Sortilin and p75 NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells , p75 NTR p75 neurotrophin receptor , Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3
    Figure Legend Snippet: Crosslinking and precipitation of p75 NTR :Sortilin:proNT3 complexes HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75 NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75 NTR , and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75 NTR . The expression of Sortilin and p75 NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells , p75 NTR p75 neurotrophin receptor , Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3

    Techniques Used: Transfection, Variant Assay, Incubation, Immunoprecipitation, SDS Page, Expressing

    Immunoblotting of inner ear tissue for Sortilin, p75 NTR , proNT3 and mature NT3 Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75 NTR , proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75 NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75 NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75 NTR anti- p75 NTR , αproNT3 anti-proNT3, αNT3 anti-NT3 , αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5
    Figure Legend Snippet: Immunoblotting of inner ear tissue for Sortilin, p75 NTR , proNT3 and mature NT3 Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75 NTR , proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75 NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75 NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75 NTR anti- p75 NTR , αproNT3 anti-proNT3, αNT3 anti-NT3 , αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5

    Techniques Used: SDS Page, Protein Concentration, Recombinant

    ProNT3 induced apoptosis in SCG neurons The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF , None no (pro)neurotrophins added , NT3 neurotrophin-3, proNT3 proform of NT3.
    Figure Legend Snippet: ProNT3 induced apoptosis in SCG neurons The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF , None no (pro)neurotrophins added , NT3 neurotrophin-3, proNT3 proform of NT3.

    Techniques Used: Incubation

    SPR analysis of ligand binding to immobilised Sortilin SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A) . This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B) , and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D) . The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C) . NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance
    Figure Legend Snippet: SPR analysis of ligand binding to immobilised Sortilin SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A) . This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B) , and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D) . The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C) . NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance

    Techniques Used: SPR Assay, Ligand Binding Assay, Sequencing

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    Alomone Labs rabbit anti kv7 1 antibody
    Mutation of phosphorylated serines does not affect subcellular localization of <t>Kv7.1.</t> Alanine ( A ) and aspartate ( B ) mutants of the identified serine phosphorylation sites were coexpressed with the ER marker pDsRed2-ER in MDCK cells. The cells were stained
    Rabbit Anti Kv7 1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs chrm3
    Repression of P2rx1 by progressive vascular endothelial damage. ( a – c ) RQ-PCR ( a ) and western blot ( b , c ) analysis of the expressions of the cholinergic receptor muscarinic 2 ( Chrm2 ), cholinergic receptor muscarinic 3 ( <t>Chrm3</t> ), and purinergic receptor P2X 1 ( P2rx1 ) genes in the bladders of rats in the indicated groups. Expression levels of the indicated transcripts are presented as % Gapdh . For western blot analysis, β-actin was used as a loading control, and the expression levels of the indicated proteins were normalized to the β-actin value. ( d , e ) Representative images ( d ) and quantification analysis ( e ) of the immunohistochemical staining for the P2rx1 protein (original magnification ×200, scale bar = 200 μm). All quantification results are presented as mean ± SEM. *p
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    Alomone Labs anti kv7 3
    Kv7.2 and 7.3 protein and mRNA expression levels were decreased in the CeA in SHRs. ( A – D ) Original gel images and quantification of band density show the total protein levels of Kv7.2 and <t>Kv7.3</t> in the CeA ( A ), PVN ( B ), HP ( C ), and PFC ( D ) from WKY rats and SHRs ( n =6 samples in each group). In these protein assays, each sample consisting of respective tissues from one rat. ( E – G ) Summary of quantitative RT–PCR data show mRNA levels of Kv7.2 and Kv7.3 in the CeA ( E ), PVN ( F ), and HP ( G ) from WKY rats and SHRs ( n =5 samples in each group). Data are presented as the mean ± SEM. * P
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    Alomone Labs rabbit anti asic3 serum
    Soma size distribution of knee joint afferents (A) was unimodal with a broad range of size and similar to that of <t>ASIC3-IR</t> knee joint afferents (C). CGRP-IR knee joint afferents consisted of small to medium cells with most cells smaller than 500μm 2 (B). ASIC3 and CGRP double labeled knee joint afferents were small to medium as well (D) whereas ASIC3 without CGRP-IR knee joint afferents were larger (E). There were no significant differences in soma size distribution among each group in knee joint afferents, CGRP, ASIC3 and ASIC3+CGRP. Only significant difference in soma size distribution was observed in ASIC3 without CGRP (E). Carrageenan-induced arthritis resulted in an overall shift in soma size distribution of ASIC3 without CGRP-IR knee joint afferents to the left (E). * p
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    Mutation of phosphorylated serines does not affect subcellular localization of Kv7.1. Alanine ( A ) and aspartate ( B ) mutants of the identified serine phosphorylation sites were coexpressed with the ER marker pDsRed2-ER in MDCK cells. The cells were stained

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Mutation of phosphorylated serines does not affect subcellular localization of Kv7.1. Alanine ( A ) and aspartate ( B ) mutants of the identified serine phosphorylation sites were coexpressed with the ER marker pDsRed2-ER in MDCK cells. The cells were stained

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Mutagenesis, Marker, Staining

    Summary of proteomics results for Kv7.1. A : topological representation of the Kv7.1 channel, including the amino acid sequence. The amino acid sequences highlighted in blue were covered in the mass spectrometry experiments, and the identified phosphorylation

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Summary of proteomics results for Kv7.1. A : topological representation of the Kv7.1 channel, including the amino acid sequence. The amino acid sequences highlighted in blue were covered in the mass spectrometry experiments, and the identified phosphorylation

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Sequencing, Mass Spectrometry

    Inhibition of PKA leads to internalization and possibly lysosomal degradation of Kv7.1.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Inhibition of PKA leads to internalization and possibly lysosomal degradation of Kv7.1.

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Inhibition

    Kv7.1 accumulates intracellularly upon exposure to PKA inhibitors KT-5720 and myristoylated protein kinase A inhibitor 14–22 amide. A : MDCK cells stably expressing Kv7.1 were subjected to a calcium switch for 24 h (control) followed by treatment

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Kv7.1 accumulates intracellularly upon exposure to PKA inhibitors KT-5720 and myristoylated protein kinase A inhibitor 14–22 amide. A : MDCK cells stably expressing Kv7.1 were subjected to a calcium switch for 24 h (control) followed by treatment

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Stable Transfection, Expressing

    Kv7.1 codistributes with late endosomal/lysosomal markers in response to H-89 treatment. A : confocal scans of MDCK-Kv7.1 cells treated with 20 μM H-89 for 3 h and colabeled with the late endosomal/lysosomal marker LAMP-2. Scale bar = 10 ( top )

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Kv7.1 codistributes with late endosomal/lysosomal markers in response to H-89 treatment. A : confocal scans of MDCK-Kv7.1 cells treated with 20 μM H-89 for 3 h and colabeled with the late endosomal/lysosomal marker LAMP-2. Scale bar = 10 ( top )

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Marker

    Small-interfering RNA (siRNA) knockdown of Nedd4-2 protects Kv7.1 from internalization upon PKA inhibition. A : MDCK cells stably expressing Kv7.1 were transiently transfected with siRNA targeting Nedd4-2 or with noncoding control siRNA. Enhanced GFP (eGFP)

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Small-interfering RNA (siRNA) knockdown of Nedd4-2 protects Kv7.1 from internalization upon PKA inhibition. A : MDCK cells stably expressing Kv7.1 were transiently transfected with siRNA targeting Nedd4-2 or with noncoding control siRNA. Enhanced GFP (eGFP)

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Small Interfering RNA, Inhibition, Stable Transfection, Expressing, Transfection

    Kv7.1-YA localization is unaffected by PKA inhibition. MDCK cells stably expressing the Kv7.1-YA mutant were treated with 20 μM H-89 for 3–5 h. A : confocal scans of MDCK-Kv7.1-YA cells before (0 h) and 3 (3 h H-89) and 5 (5 h H-89) h after

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Kv7.1-YA localization is unaffected by PKA inhibition. MDCK cells stably expressing the Kv7.1-YA mutant were treated with 20 μM H-89 for 3–5 h. A : confocal scans of MDCK-Kv7.1-YA cells before (0 h) and 3 (3 h H-89) and 5 (5 h H-89) h after

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Inhibition, Stable Transfection, Expressing, Mutagenesis

    Kv7.1 redistribution is not caused by increased Nedd4-2 binding or ubiquitinylation. A : representative Western blot showing the effects of 20 μM H-89 (3 h exposure) and 50 μM forskolin (1 h exposure) on the abundance of S 468 -phosphorylated

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Kv7.1 redistribution is not caused by increased Nedd4-2 binding or ubiquitinylation. A : representative Western blot showing the effects of 20 μM H-89 (3 h exposure) and 50 μM forskolin (1 h exposure) on the abundance of S 468 -phosphorylated

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Binding Assay, Western Blot

    Kv7.1 accumulates intracellularly upon inhibition of protein kinase A (PKA). Madin-Darby canine kidney (MDCK) cells stably expressing Kv7.1 were subjected to a calcium switch for 24 h followed by treatment with 20 μM H-89 for 3–5 h. A

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Kv7.1 accumulates intracellularly upon inhibition of protein kinase A (PKA). Madin-Darby canine kidney (MDCK) cells stably expressing Kv7.1 were subjected to a calcium switch for 24 h followed by treatment with 20 μM H-89 for 3–5 h. A

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Inhibition, Stable Transfection, Expressing

    Intracellular accumulation in response to PKA inhibition is specific for Kv7.1. MDCK cells were transfected with Kv7.4 and allowed to polarize before being subjected to either 20 μM H-89 or 10 μM KT-5720 treatments for 3 h. As demonstrated,

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Intracellular accumulation in response to PKA inhibition is specific for Kv7.1. MDCK cells were transfected with Kv7.4 and allowed to polarize before being subjected to either 20 μM H-89 or 10 μM KT-5720 treatments for 3 h. As demonstrated,

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Inhibition, Transfection

    PKA activation promotes Kv7.1 surface expression in polarized MDCK cells. MDCK cells stably expressing Kv7.1 were grown to confluence and allowed to polarize. The cells were then treated with 50 μM forskolin for 1 h. Cells were fixed before and

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: PKA activation promotes Kv7.1 surface expression in polarized MDCK cells. MDCK cells stably expressing Kv7.1 were grown to confluence and allowed to polarize. The cells were then treated with 50 μM forskolin for 1 h. Cells were fixed before and

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Activation Assay, Expressing, Stable Transfection

    Mimicked phosphorylation of T470 results in reduced membrane expression of Kv7.1, but Kv7.1-T470A is still endocytosed in response to PKA inhibition. Kv7.1-T470A and Kv7.1-T470D mutants were coexpressed with DsRed2-ER in MDCK cells, which were allowed

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    doi: 10.1152/ajpcell.00383.2014

    Figure Lengend Snippet: Mimicked phosphorylation of T470 results in reduced membrane expression of Kv7.1, but Kv7.1-T470A is still endocytosed in response to PKA inhibition. Kv7.1-T470A and Kv7.1-T470D mutants were coexpressed with DsRed2-ER in MDCK cells, which were allowed

    Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

    Techniques: Expressing, Inhibition

    Repression of P2rx1 by progressive vascular endothelial damage. ( a – c ) RQ-PCR ( a ) and western blot ( b , c ) analysis of the expressions of the cholinergic receptor muscarinic 2 ( Chrm2 ), cholinergic receptor muscarinic 3 ( Chrm3 ), and purinergic receptor P2X 1 ( P2rx1 ) genes in the bladders of rats in the indicated groups. Expression levels of the indicated transcripts are presented as % Gapdh . For western blot analysis, β-actin was used as a loading control, and the expression levels of the indicated proteins were normalized to the β-actin value. ( d , e ) Representative images ( d ) and quantification analysis ( e ) of the immunohistochemical staining for the P2rx1 protein (original magnification ×200, scale bar = 200 μm). All quantification results are presented as mean ± SEM. *p

    Journal: Scientific Reports

    Article Title: Induction of detrusor underactivity by extensive vascular endothelial damages of iliac arteries in a rat model and its pathophysiology in the genetic levels

    doi: 10.1038/s41598-019-52811-4

    Figure Lengend Snippet: Repression of P2rx1 by progressive vascular endothelial damage. ( a – c ) RQ-PCR ( a ) and western blot ( b , c ) analysis of the expressions of the cholinergic receptor muscarinic 2 ( Chrm2 ), cholinergic receptor muscarinic 3 ( Chrm3 ), and purinergic receptor P2X 1 ( P2rx1 ) genes in the bladders of rats in the indicated groups. Expression levels of the indicated transcripts are presented as % Gapdh . For western blot analysis, β-actin was used as a loading control, and the expression levels of the indicated proteins were normalized to the β-actin value. ( d , e ) Representative images ( d ) and quantification analysis ( e ) of the immunohistochemical staining for the P2rx1 protein (original magnification ×200, scale bar = 200 μm). All quantification results are presented as mean ± SEM. *p

    Article Snippet: Protein levels were assessed from 50 μg of extracts separated using 10% SDS-PAGE gels by probing with antibodies specific to P2rx1 (APR-001, Alomone Labs), Chrm2 (AMR-002, Alomone Labs,), Chrm3 (AMR-006, Alomone Labs, Israel), α-SMA (ab5694, Rabbit, Abcam), vimentin (sc-6260, Santa Cruz Biotechnology Inc., CA), and β-actin (Sigma-Aldrich).

    Techniques: Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemistry, Staining

    Kv7.2 and 7.3 protein and mRNA expression levels were decreased in the CeA in SHRs. ( A – D ) Original gel images and quantification of band density show the total protein levels of Kv7.2 and Kv7.3 in the CeA ( A ), PVN ( B ), HP ( C ), and PFC ( D ) from WKY rats and SHRs ( n =6 samples in each group). In these protein assays, each sample consisting of respective tissues from one rat. ( E – G ) Summary of quantitative RT–PCR data show mRNA levels of Kv7.2 and Kv7.3 in the CeA ( E ), PVN ( F ), and HP ( G ) from WKY rats and SHRs ( n =5 samples in each group). Data are presented as the mean ± SEM. * P

    Journal: Cardiovascular Research

    Article Title: Impaired Kv7 channel activity in the central amygdala contributes to elevated sympathetic outflow in hypertension

    doi: 10.1093/cvr/cvab031

    Figure Lengend Snippet: Kv7.2 and 7.3 protein and mRNA expression levels were decreased in the CeA in SHRs. ( A – D ) Original gel images and quantification of band density show the total protein levels of Kv7.2 and Kv7.3 in the CeA ( A ), PVN ( B ), HP ( C ), and PFC ( D ) from WKY rats and SHRs ( n =6 samples in each group). In these protein assays, each sample consisting of respective tissues from one rat. ( E – G ) Summary of quantitative RT–PCR data show mRNA levels of Kv7.2 and Kv7.3 in the CeA ( E ), PVN ( F ), and HP ( G ) from WKY rats and SHRs ( n =5 samples in each group). Data are presented as the mean ± SEM. * P

    Article Snippet: Immunohistochemical staining was performed to determine the spatial distribution of Kv7 channels in the CeA–RVLM neurons by using anti-Kv7.3 (Alomone Labs) antibody.

    Techniques: Expressing, Quantitative RT-PCR

    Reduced expression of Kv7.2/Kv7.3 in the CeA was independent of high blood pressure in SHRs. ( A and B ) Western blot images and quantification of band density show the total protein levels of Kv7.2 ( A ) and Kv7.3 ( B ) (normalized to β-actin) in the CeA in 4, 7, and 13 weeks old WKY rats and SHRs ( n =4 rats in each group). ( C ) Summary data show mean ABP measured by telemetry approach in SHRs subjected to the CGx and sham surgery ( n =6 rats in each group). (D–F) Western blot images and quantification of band density show the total protein levels of Kv7.2 and Kv7.3 (normalized to β-actin) in the CeA ( D ), the PVN ( E ), and HP ( F ) in SHRs subjected to CGx or sham surgery ( n =6 rats in each group). Data are presented as the mean±SEM. * P

    Journal: Cardiovascular Research

    Article Title: Impaired Kv7 channel activity in the central amygdala contributes to elevated sympathetic outflow in hypertension

    doi: 10.1093/cvr/cvab031

    Figure Lengend Snippet: Reduced expression of Kv7.2/Kv7.3 in the CeA was independent of high blood pressure in SHRs. ( A and B ) Western blot images and quantification of band density show the total protein levels of Kv7.2 ( A ) and Kv7.3 ( B ) (normalized to β-actin) in the CeA in 4, 7, and 13 weeks old WKY rats and SHRs ( n =4 rats in each group). ( C ) Summary data show mean ABP measured by telemetry approach in SHRs subjected to the CGx and sham surgery ( n =6 rats in each group). (D–F) Western blot images and quantification of band density show the total protein levels of Kv7.2 and Kv7.3 (normalized to β-actin) in the CeA ( D ), the PVN ( E ), and HP ( F ) in SHRs subjected to CGx or sham surgery ( n =6 rats in each group). Data are presented as the mean±SEM. * P

    Article Snippet: Immunohistochemical staining was performed to determine the spatial distribution of Kv7 channels in the CeA–RVLM neurons by using anti-Kv7.3 (Alomone Labs) antibody.

    Techniques: Expressing, Western Blot

    M-currents were decreased in CeA–RVLM neurons in SHRs. ( A ) Images show retrograde tracer injection sites in the RVLM viewed by light (a) and fluorescent (b) microscope and Fluosphere labelled neurons in the CeA viewed by light (c and e) and fluorescent (d and f) microscope. ( B ) Immunostaining images show that Kv7.3 immunoreactivities were expressed on DiI-labelled CeA neurons. ( C and D ) Original recordings ( C ) and summary data ( D ) show the basal and QO-58-induced M-currents, which were defined as XE-991-sensitive tail currents in basal and the presence of QO-58, were significantly diminished in retrogradely labelled CeA–RVLM neurons in SHRs ( n =10 neurons in 4 rats) compared with WKY rats ( n =9 neurons in 4 rats).*** P

    Journal: Cardiovascular Research

    Article Title: Impaired Kv7 channel activity in the central amygdala contributes to elevated sympathetic outflow in hypertension

    doi: 10.1093/cvr/cvab031

    Figure Lengend Snippet: M-currents were decreased in CeA–RVLM neurons in SHRs. ( A ) Images show retrograde tracer injection sites in the RVLM viewed by light (a) and fluorescent (b) microscope and Fluosphere labelled neurons in the CeA viewed by light (c and e) and fluorescent (d and f) microscope. ( B ) Immunostaining images show that Kv7.3 immunoreactivities were expressed on DiI-labelled CeA neurons. ( C and D ) Original recordings ( C ) and summary data ( D ) show the basal and QO-58-induced M-currents, which were defined as XE-991-sensitive tail currents in basal and the presence of QO-58, were significantly diminished in retrogradely labelled CeA–RVLM neurons in SHRs ( n =10 neurons in 4 rats) compared with WKY rats ( n =9 neurons in 4 rats).*** P

    Article Snippet: Immunohistochemical staining was performed to determine the spatial distribution of Kv7 channels in the CeA–RVLM neurons by using anti-Kv7.3 (Alomone Labs) antibody.

    Techniques: Injection, Microscopy, Immunostaining

    Soma size distribution of knee joint afferents (A) was unimodal with a broad range of size and similar to that of ASIC3-IR knee joint afferents (C). CGRP-IR knee joint afferents consisted of small to medium cells with most cells smaller than 500μm 2 (B). ASIC3 and CGRP double labeled knee joint afferents were small to medium as well (D) whereas ASIC3 without CGRP-IR knee joint afferents were larger (E). There were no significant differences in soma size distribution among each group in knee joint afferents, CGRP, ASIC3 and ASIC3+CGRP. Only significant difference in soma size distribution was observed in ASIC3 without CGRP (E). Carrageenan-induced arthritis resulted in an overall shift in soma size distribution of ASIC3 without CGRP-IR knee joint afferents to the left (E). * p

    Journal: The journal of pain : official journal of the American Pain Society

    Article Title: Acid sensing ion channel 3 expression in mice knee joint afferents and effects of carrageenan-induced arthritis

    doi: 10.1016/j.jpain.2008.10.010

    Figure Lengend Snippet: Soma size distribution of knee joint afferents (A) was unimodal with a broad range of size and similar to that of ASIC3-IR knee joint afferents (C). CGRP-IR knee joint afferents consisted of small to medium cells with most cells smaller than 500μm 2 (B). ASIC3 and CGRP double labeled knee joint afferents were small to medium as well (D) whereas ASIC3 without CGRP-IR knee joint afferents were larger (E). There were no significant differences in soma size distribution among each group in knee joint afferents, CGRP, ASIC3 and ASIC3+CGRP. Only significant difference in soma size distribution was observed in ASIC3 without CGRP (E). Carrageenan-induced arthritis resulted in an overall shift in soma size distribution of ASIC3 without CGRP-IR knee joint afferents to the left (E). * p

    Article Snippet: The primary antisera used in this study were rabbit anti-ASIC3 serum (1:500; Alomone Labs, Jerusalem, Israel) and rabbit anti-CGRP serum (1:1000; Peninsula Laboratories, SanCalros, CA).

    Techniques: Labeling

    The number of knee joint afferents (A) and the percentage of knee joint afferents immunoreactive for CGRP (B), ASIC3 (C), ASIC3+CGRP (D) and ASIC3 without CGRP (E). Values are represented as mean±SEM. Knee joint afferents immunoreactive for CGRP, ASIC3 and ASIC3+CGRP were significantly increased 24h after carrageenan-induced arthritis. * p

    Journal: The journal of pain : official journal of the American Pain Society

    Article Title: Acid sensing ion channel 3 expression in mice knee joint afferents and effects of carrageenan-induced arthritis

    doi: 10.1016/j.jpain.2008.10.010

    Figure Lengend Snippet: The number of knee joint afferents (A) and the percentage of knee joint afferents immunoreactive for CGRP (B), ASIC3 (C), ASIC3+CGRP (D) and ASIC3 without CGRP (E). Values are represented as mean±SEM. Knee joint afferents immunoreactive for CGRP, ASIC3 and ASIC3+CGRP were significantly increased 24h after carrageenan-induced arthritis. * p

    Article Snippet: The primary antisera used in this study were rabbit anti-ASIC3 serum (1:500; Alomone Labs, Jerusalem, Israel) and rabbit anti-CGRP serum (1:1000; Peninsula Laboratories, SanCalros, CA).

    Techniques:

    Fast Blue labeling (A,B,C and D) and immnohistochemistry staining for ASIC3 (E,F,G and H) and CGRP (I,J,K and L) showing DRG from ASIC3 +/+ (first and second rows) and ASIC3 -/- (third and fourth rows) mice without joint inflammation (first and third rows) and those 24h after joint inflammation (second and fourth rows). Photos in each row are the same DRG. Arrows indicate Fast Blue labeled DRG neurons. Asterisks indicate ASIC3-IR neurons. No immunoreactivity for ASIC3 was observed in ASIC3 -/-mice(G and H), while CGRP was observed in all groups. Some DRG neurons showing both ASIC3 and CGRP immunoreactivity were observed as yellow in ASIC3+CGRP merged images (M and N).

    Journal: The journal of pain : official journal of the American Pain Society

    Article Title: Acid sensing ion channel 3 expression in mice knee joint afferents and effects of carrageenan-induced arthritis

    doi: 10.1016/j.jpain.2008.10.010

    Figure Lengend Snippet: Fast Blue labeling (A,B,C and D) and immnohistochemistry staining for ASIC3 (E,F,G and H) and CGRP (I,J,K and L) showing DRG from ASIC3 +/+ (first and second rows) and ASIC3 -/- (third and fourth rows) mice without joint inflammation (first and third rows) and those 24h after joint inflammation (second and fourth rows). Photos in each row are the same DRG. Arrows indicate Fast Blue labeled DRG neurons. Asterisks indicate ASIC3-IR neurons. No immunoreactivity for ASIC3 was observed in ASIC3 -/-mice(G and H), while CGRP was observed in all groups. Some DRG neurons showing both ASIC3 and CGRP immunoreactivity were observed as yellow in ASIC3+CGRP merged images (M and N).

    Article Snippet: The primary antisera used in this study were rabbit anti-ASIC3 serum (1:500; Alomone Labs, Jerusalem, Israel) and rabbit anti-CGRP serum (1:1000; Peninsula Laboratories, SanCalros, CA).

    Techniques: Labeling, Staining, Mouse Assay