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
    Price from $9.99 to $1999.99
    rabbit anti nt3 - by Bioz Stars, 2022-08
    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 rabbit anti p2x3r
    Go6976 reduces α,β-meATP–elicited flinch nocifensive responses in complete Freund adjuvant–treated rats, and PKCβ1 is not expressed in <t>P2X3R-containing</t> dorsal root ganglia (DRG) neurons. (A) In saline control rats,
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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
    Chrm3, 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 anti clc 3 ab
    <t>Anti-ClC-3</t> Ab dialysis abolishes expressed I gpClC-3 in NIH/3T3 cells A , phase contrast (left panel) and fluorescence micrographs (right panel) of NIH/3T3 cell transfected with gpClC-3-GFP. B , representative I gpClC-3 recorded from GFP-positive NIH/3T3 cells (as shown in A ) under isotonic, hypotonic and hypertonic conditions over the range -100 to +120 mV. C , effects of anti-ClC-3 Ab on I gpClC-3 at ±80 mV when pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). D , mean current densities recorded from untransfected NIH/3T3 cells (blue, n = 38), gpClC-3-GFP-transfected cells dialysed with standard intracellular pipette solutions (green, n = 9), preabsorbed anti-ClC-3 Ab (black, n = 4), and anti-ClC-3 Ab alone (red, n = 9).
    Anti Clc 3 Ab, 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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    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

    Go6976 reduces α,β-meATP–elicited flinch nocifensive responses in complete Freund adjuvant–treated rats, and PKCβ1 is not expressed in P2X3R-containing dorsal root ganglia (DRG) neurons. (A) In saline control rats,

    Journal: Pain

    Article Title: Epac–protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation

    doi: 10.1097/j.pain.0000000000000547

    Figure Lengend Snippet: Go6976 reduces α,β-meATP–elicited flinch nocifensive responses in complete Freund adjuvant–treated rats, and PKCβ1 is not expressed in P2X3R-containing dorsal root ganglia (DRG) neurons. (A) In saline control rats,

    Article Snippet: Antibodies used were mouse anti-Epac1, anti-Epac2 (1:1000; Cell Signaling, Danvers, MA), mouse anti-PKCε (1:1000; Santa Cruz), rabbit anti-pPKCε (1:1000; Santa Cruz), and rabbit anti-P2X3R (1:2000; Alomone Labs). β-Actin, probed with anti-β-actin (1:10,000; Chemicon, Temeculla, CA), or the neuronal marker, β-tubulin, probed with anti β-tubulin antibody (1:2000; Santa Cruz), was used as loading control.

    Techniques:

    Phosphorylated PKC (pPKC) isoform and P2X3R expression in dorsal root ganglia slices prepared from complete Freund adjuvant–treated rats. Upper: pPKCα and P2X3Rs were expressed only in small or medium cells. pPKCα labels were found

    Journal: Pain

    Article Title: Epac–protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation

    doi: 10.1097/j.pain.0000000000000547

    Figure Lengend Snippet: Phosphorylated PKC (pPKC) isoform and P2X3R expression in dorsal root ganglia slices prepared from complete Freund adjuvant–treated rats. Upper: pPKCα and P2X3Rs were expressed only in small or medium cells. pPKCα labels were found

    Article Snippet: Antibodies used were mouse anti-Epac1, anti-Epac2 (1:1000; Cell Signaling, Danvers, MA), mouse anti-PKCε (1:1000; Santa Cruz), rabbit anti-pPKCε (1:1000; Santa Cruz), and rabbit anti-P2X3R (1:2000; Alomone Labs). β-Actin, probed with anti-β-actin (1:10,000; Chemicon, Temeculla, CA), or the neuronal marker, β-tubulin, probed with anti β-tubulin antibody (1:2000; Santa Cruz), was used as loading control.

    Techniques: Expressing

    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

    Anti-ClC-3 Ab dialysis abolishes expressed I gpClC-3 in NIH/3T3 cells A , phase contrast (left panel) and fluorescence micrographs (right panel) of NIH/3T3 cell transfected with gpClC-3-GFP. B , representative I gpClC-3 recorded from GFP-positive NIH/3T3 cells (as shown in A ) under isotonic, hypotonic and hypertonic conditions over the range -100 to +120 mV. C , effects of anti-ClC-3 Ab on I gpClC-3 at ±80 mV when pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). D , mean current densities recorded from untransfected NIH/3T3 cells (blue, n = 38), gpClC-3-GFP-transfected cells dialysed with standard intracellular pipette solutions (green, n = 9), preabsorbed anti-ClC-3 Ab (black, n = 4), and anti-ClC-3 Ab alone (red, n = 9).

    Journal: The Journal of Physiology

    Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

    doi: 10.1111/j.1469-7793.2001.0437i.x

    Figure Lengend Snippet: Anti-ClC-3 Ab dialysis abolishes expressed I gpClC-3 in NIH/3T3 cells A , phase contrast (left panel) and fluorescence micrographs (right panel) of NIH/3T3 cell transfected with gpClC-3-GFP. B , representative I gpClC-3 recorded from GFP-positive NIH/3T3 cells (as shown in A ) under isotonic, hypotonic and hypertonic conditions over the range -100 to +120 mV. C , effects of anti-ClC-3 Ab on I gpClC-3 at ±80 mV when pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). D , mean current densities recorded from untransfected NIH/3T3 cells (blue, n = 38), gpClC-3-GFP-transfected cells dialysed with standard intracellular pipette solutions (green, n = 9), preabsorbed anti-ClC-3 Ab (black, n = 4), and anti-ClC-3 Ab alone (red, n = 9).

    Article Snippet: We first demonstrate that intracellular dialysis with a commercially available antibody which targets a specific 70 amino acid epitope on the carboxyl terminus of rat ClC-3 (anti-ClC-3 Ab) abolishes expressed I gpClC-3 in transfected NIH/3T3 cells, and then demonstrate that intracellular dialysis or injection of anti-ClC-3 Ab into native cardiac and vascular smooth muscle myocytes and Xenopus oocytes abolishes or significantly attenuates native VSOAC function.

    Techniques: Fluorescence, Transfection, Transferring

    Anti-ClC-3 Ab abolishes native VSOAC currents in canine PASMCs A , time course of VSOAC currents from two PASMCs dialysed with either anti-ClC-3 Ab (5 μg ml −1 ) preabsorbed with antigen (50 μg ml −1 ; ○), or anti-ClC-3 Ab alone (5 μg ml −1 ; •). Inset: Western blot analysis of native ClC-3 expression in isolated canine PASMCs. B , mean current densities in cells dialysed with either standard intracellular solutions ( n = 11), preabsorbed anti-ClC-3 Ab ( n = 4) or anti-ClC-3 Ab alone ( n = 5).

    Journal: The Journal of Physiology

    Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

    doi: 10.1111/j.1469-7793.2001.0437i.x

    Figure Lengend Snippet: Anti-ClC-3 Ab abolishes native VSOAC currents in canine PASMCs A , time course of VSOAC currents from two PASMCs dialysed with either anti-ClC-3 Ab (5 μg ml −1 ) preabsorbed with antigen (50 μg ml −1 ; ○), or anti-ClC-3 Ab alone (5 μg ml −1 ; •). Inset: Western blot analysis of native ClC-3 expression in isolated canine PASMCs. B , mean current densities in cells dialysed with either standard intracellular solutions ( n = 11), preabsorbed anti-ClC-3 Ab ( n = 4) or anti-ClC-3 Ab alone ( n = 5).

    Article Snippet: We first demonstrate that intracellular dialysis with a commercially available antibody which targets a specific 70 amino acid epitope on the carboxyl terminus of rat ClC-3 (anti-ClC-3 Ab) abolishes expressed I gpClC-3 in transfected NIH/3T3 cells, and then demonstrate that intracellular dialysis or injection of anti-ClC-3 Ab into native cardiac and vascular smooth muscle myocytes and Xenopus oocytes abolishes or significantly attenuates native VSOAC function.

    Techniques: Western Blot, Expressing, Isolation

    Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions. Aa , pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). Ab , mean current densities for preabsorbed anti-ClC-3 Ab (□, n = 3) or anti-ClC-3 Ab alone (▪, n = 5) at times indicated. B, same as A , except cells were preswelled by pre-exposure to hypotonic bath solutions prior to membrane rupture and dialysis; preabsorbed anti-ClC-3 Ab (□, n = 4), and anti-ClC-3 Ab alone (▪, n = 5).

    Journal: The Journal of Physiology

    Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

    doi: 10.1111/j.1469-7793.2001.0437i.x

    Figure Lengend Snippet: Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions. Aa , pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). Ab , mean current densities for preabsorbed anti-ClC-3 Ab (□, n = 3) or anti-ClC-3 Ab alone (▪, n = 5) at times indicated. B, same as A , except cells were preswelled by pre-exposure to hypotonic bath solutions prior to membrane rupture and dialysis; preabsorbed anti-ClC-3 Ab (□, n = 4), and anti-ClC-3 Ab alone (▪, n = 5).

    Article Snippet: We first demonstrate that intracellular dialysis with a commercially available antibody which targets a specific 70 amino acid epitope on the carboxyl terminus of rat ClC-3 (anti-ClC-3 Ab) abolishes expressed I gpClC-3 in transfected NIH/3T3 cells, and then demonstrate that intracellular dialysis or injection of anti-ClC-3 Ab into native cardiac and vascular smooth muscle myocytes and Xenopus oocytes abolishes or significantly attenuates native VSOAC function.

    Techniques: Transferring

    Anti-ClC-3 Ab injection inhibits native VSOAC currents in Xenopus laevis oocytes A , Western blot analysis of native ClC-3 expression in oocytes. B , 4-phorbol 12,13-dibutyrate (PDBu) inhibition of native VSOAC currents in oocytes (-100 to +120 mV). C, time course of VSOAC activation in response to hypotonic bath solutions from a non-injected control oocyte (○) and an oocyte injected with anti-ClC-3 Ab (15 μg ml -1 ; •) at time shown. Cells were held at -30 mV for 30 ms, hyperpolarized to -100 mV for 210 ms and then depolarized to +100 mV for 210 ms, repetitively at 2 Hz. D, mean current densities from control oocytes ( n = 10), oocytes injected with anti-ClC-3 Ab alone (15 μg ml -1 ) ( n = 4) or injected with preabsorbed anti-ClC-3 Ab (150 μg ml -1 ) ( n = 4). Peak current densities were measured after 5 min exposure to isotonic solutions, and after 85 min exposure to hypotonic solutions.

    Journal: The Journal of Physiology

    Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

    doi: 10.1111/j.1469-7793.2001.0437i.x

    Figure Lengend Snippet: Anti-ClC-3 Ab injection inhibits native VSOAC currents in Xenopus laevis oocytes A , Western blot analysis of native ClC-3 expression in oocytes. B , 4-phorbol 12,13-dibutyrate (PDBu) inhibition of native VSOAC currents in oocytes (-100 to +120 mV). C, time course of VSOAC activation in response to hypotonic bath solutions from a non-injected control oocyte (○) and an oocyte injected with anti-ClC-3 Ab (15 μg ml -1 ; •) at time shown. Cells were held at -30 mV for 30 ms, hyperpolarized to -100 mV for 210 ms and then depolarized to +100 mV for 210 ms, repetitively at 2 Hz. D, mean current densities from control oocytes ( n = 10), oocytes injected with anti-ClC-3 Ab alone (15 μg ml -1 ) ( n = 4) or injected with preabsorbed anti-ClC-3 Ab (150 μg ml -1 ) ( n = 4). Peak current densities were measured after 5 min exposure to isotonic solutions, and after 85 min exposure to hypotonic solutions.

    Article Snippet: We first demonstrate that intracellular dialysis with a commercially available antibody which targets a specific 70 amino acid epitope on the carboxyl terminus of rat ClC-3 (anti-ClC-3 Ab) abolishes expressed I gpClC-3 in transfected NIH/3T3 cells, and then demonstrate that intracellular dialysis or injection of anti-ClC-3 Ab into native cardiac and vascular smooth muscle myocytes and Xenopus oocytes abolishes or significantly attenuates native VSOAC function.

    Techniques: Injection, Western Blot, Expressing, Inhibition, Activation Assay, Mass Spectrometry