kv2 1 c terminus  (Alomone Labs)


Bioz Verified Symbol Alomone Labs is a verified supplier
Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 93
      Buy from Supplier

    Structured Review

    Alomone Labs kv2 1 c terminus
    A. A schematic presentation of the <t>Kv2.1</t> channel showing fragments generated as GST fusion proteins. The grey box on the left, the 6 dark boxes and the grey box on the right denote the N terminus, the 6 trans membrane and the C terminus domains, respectively. Numbers denote amino acids. B. GST fusion proteins corresponding to Kv2.1 C terminus but not N terminus pull down syntaxin from PC12 cell lysate. Proteins (each at 150 pmol) immobilized on glutathione affinity beads were incubated with 10 6 cells in K-Glu buffer with 1% Triton X-100 for 12 hr at 4°C. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) and immunobloted with syntaxin antibody ( IB Syx ), or stained with Ponceau S (lower panel). PC12 lysates (0.5% of that processed in the pull down reaction) were loaded for reference. Right and left panels are two separate experiments. Molecular weights are marked on the right. C. GST-fused Kv2.1-C1a protein or GST alone (150 pmole each) immobilized on glutathione affinity beads were incubated with 35 S-labeled synaptotagmin ( Syt ), 35 S-labeled syntaxin ( Syx ), 35 S-labeled SNAP-25 or 35 S-labeled VAMP2 for 1 h. in 1 ml 0.1% Triton X-100. Gluthation-eluted proteins ( right panel ) and input (20 µl out of 1000 µl of reaction mixture taken before the addition of beads; left panel ) were analyzed by SDS-PAGE.
    Kv2 1 C Terminus, 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
    https://www.bioz.com/result/kv2 1 c terminus/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kv2 1 c terminus - by Bioz Stars, 2023-11
    93/100 stars

    Images

    1) Product Images from "Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells"

    Article Title: Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001381

    A. A schematic presentation of the Kv2.1 channel showing fragments generated as GST fusion proteins. The grey box on the left, the 6 dark boxes and the grey box on the right denote the N terminus, the 6 trans membrane and the C terminus domains, respectively. Numbers denote amino acids. B. GST fusion proteins corresponding to Kv2.1 C terminus but not N terminus pull down syntaxin from PC12 cell lysate. Proteins (each at 150 pmol) immobilized on glutathione affinity beads were incubated with 10 6 cells in K-Glu buffer with 1% Triton X-100 for 12 hr at 4°C. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) and immunobloted with syntaxin antibody ( IB Syx ), or stained with Ponceau S (lower panel). PC12 lysates (0.5% of that processed in the pull down reaction) were loaded for reference. Right and left panels are two separate experiments. Molecular weights are marked on the right. C. GST-fused Kv2.1-C1a protein or GST alone (150 pmole each) immobilized on glutathione affinity beads were incubated with 35 S-labeled synaptotagmin ( Syt ), 35 S-labeled syntaxin ( Syx ), 35 S-labeled SNAP-25 or 35 S-labeled VAMP2 for 1 h. in 1 ml 0.1% Triton X-100. Gluthation-eluted proteins ( right panel ) and input (20 µl out of 1000 µl of reaction mixture taken before the addition of beads; left panel ) were analyzed by SDS-PAGE.
    Figure Legend Snippet: A. A schematic presentation of the Kv2.1 channel showing fragments generated as GST fusion proteins. The grey box on the left, the 6 dark boxes and the grey box on the right denote the N terminus, the 6 trans membrane and the C terminus domains, respectively. Numbers denote amino acids. B. GST fusion proteins corresponding to Kv2.1 C terminus but not N terminus pull down syntaxin from PC12 cell lysate. Proteins (each at 150 pmol) immobilized on glutathione affinity beads were incubated with 10 6 cells in K-Glu buffer with 1% Triton X-100 for 12 hr at 4°C. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) and immunobloted with syntaxin antibody ( IB Syx ), or stained with Ponceau S (lower panel). PC12 lysates (0.5% of that processed in the pull down reaction) were loaded for reference. Right and left panels are two separate experiments. Molecular weights are marked on the right. C. GST-fused Kv2.1-C1a protein or GST alone (150 pmole each) immobilized on glutathione affinity beads were incubated with 35 S-labeled synaptotagmin ( Syt ), 35 S-labeled syntaxin ( Syx ), 35 S-labeled SNAP-25 or 35 S-labeled VAMP2 for 1 h. in 1 ml 0.1% Triton X-100. Gluthation-eluted proteins ( right panel ) and input (20 µl out of 1000 µl of reaction mixture taken before the addition of beads; left panel ) were analyzed by SDS-PAGE.

    Techniques Used: Generated, Incubation, SDS Page, Staining, Labeling

    (A) PC12 cells preloaded with [ 3 H]-NE and gently homogenized ( “cracked” cells), either underwent 15 min incubation in the presence of 2 mM MgATP and brain cytosol at 30°C (“priming”) or not, followed by either 15 min incubation with added 1.6 mM Ca 2+ (1 µM free Ca 2+ ; at 30° (“triggering”) or not, as indicated below the bars. Cells were pelleted and the secreted [ 3 H]-NE in the supernatant was quantified by scintillation counting and expressed as a percentage of the total [ 3 H]-NE in the cell. 10 6 cells per reaction were used. The difference between the amounts of [ 3 H]-NE secreted in the two reactions on the two bars on the right was defined and is referred to hereafter as the Ca 2+ -triggered release. (B) Cracked cells preloaded with [ 3 H]-NE underwent priming and triggering reactions in the absence or presence of 10–20 µM GST-fusion peptides corresponding to cytosolic parts of Kv2.1 and Kv1.1 (shown in ) or to GST itself (as indicated below bars). Values of Ca 2+ -triggered release measured in the presence of the peptides (see ) were normalized to the control release determined in the absence of peptides (defined as 100%). Each bar in A and B depicts the mean±s.e.m from several independent experiments (numbers of experiments are in parentheses above bars). **, p <0.001 (compared with GST). (C) Cells were stimulated to release NE in the presence of increasing concentrations of GST-fused Kv2.1-C1 protein. Each point in the curve represents mean±s.e.m values from several independent experiments (numbers of experiments in parentheses above bars). **, p<0.001 (compared with 10 nM GST-C1).
    Figure Legend Snippet: (A) PC12 cells preloaded with [ 3 H]-NE and gently homogenized ( “cracked” cells), either underwent 15 min incubation in the presence of 2 mM MgATP and brain cytosol at 30°C (“priming”) or not, followed by either 15 min incubation with added 1.6 mM Ca 2+ (1 µM free Ca 2+ ; at 30° (“triggering”) or not, as indicated below the bars. Cells were pelleted and the secreted [ 3 H]-NE in the supernatant was quantified by scintillation counting and expressed as a percentage of the total [ 3 H]-NE in the cell. 10 6 cells per reaction were used. The difference between the amounts of [ 3 H]-NE secreted in the two reactions on the two bars on the right was defined and is referred to hereafter as the Ca 2+ -triggered release. (B) Cracked cells preloaded with [ 3 H]-NE underwent priming and triggering reactions in the absence or presence of 10–20 µM GST-fusion peptides corresponding to cytosolic parts of Kv2.1 and Kv1.1 (shown in ) or to GST itself (as indicated below bars). Values of Ca 2+ -triggered release measured in the presence of the peptides (see ) were normalized to the control release determined in the absence of peptides (defined as 100%). Each bar in A and B depicts the mean±s.e.m from several independent experiments (numbers of experiments are in parentheses above bars). **, p <0.001 (compared with GST). (C) Cells were stimulated to release NE in the presence of increasing concentrations of GST-fused Kv2.1-C1 protein. Each point in the curve represents mean±s.e.m values from several independent experiments (numbers of experiments in parentheses above bars). **, p<0.001 (compared with 10 nM GST-C1).

    Techniques Used: Incubation

    kv2 1 c terminus  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 93
      Buy from Supplier

    Structured Review

    Alomone Labs kv2 1 c terminus
    A. A schematic presentation of the <t>Kv2.1</t> channel showing fragments generated as GST fusion proteins. The grey box on the left, the 6 dark boxes and the grey box on the right denote the N terminus, the 6 trans membrane and the C terminus domains, respectively. Numbers denote amino acids. B. GST fusion proteins corresponding to Kv2.1 C terminus but not N terminus pull down syntaxin from PC12 cell lysate. Proteins (each at 150 pmol) immobilized on glutathione affinity beads were incubated with 10 6 cells in K-Glu buffer with 1% Triton X-100 for 12 hr at 4°C. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) and immunobloted with syntaxin antibody ( IB Syx ), or stained with Ponceau S (lower panel). PC12 lysates (0.5% of that processed in the pull down reaction) were loaded for reference. Right and left panels are two separate experiments. Molecular weights are marked on the right. C. GST-fused Kv2.1-C1a protein or GST alone (150 pmole each) immobilized on glutathione affinity beads were incubated with 35 S-labeled synaptotagmin ( Syt ), 35 S-labeled syntaxin ( Syx ), 35 S-labeled SNAP-25 or 35 S-labeled VAMP2 for 1 h. in 1 ml 0.1% Triton X-100. Gluthation-eluted proteins ( right panel ) and input (20 µl out of 1000 µl of reaction mixture taken before the addition of beads; left panel ) were analyzed by SDS-PAGE.
    Kv2 1 C Terminus, 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
    https://www.bioz.com/result/kv2 1 c terminus/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kv2 1 c terminus - by Bioz Stars, 2023-11
    93/100 stars

    Images

    1) Product Images from "Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells"

    Article Title: Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001381

    A. A schematic presentation of the Kv2.1 channel showing fragments generated as GST fusion proteins. The grey box on the left, the 6 dark boxes and the grey box on the right denote the N terminus, the 6 trans membrane and the C terminus domains, respectively. Numbers denote amino acids. B. GST fusion proteins corresponding to Kv2.1 C terminus but not N terminus pull down syntaxin from PC12 cell lysate. Proteins (each at 150 pmol) immobilized on glutathione affinity beads were incubated with 10 6 cells in K-Glu buffer with 1% Triton X-100 for 12 hr at 4°C. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) and immunobloted with syntaxin antibody ( IB Syx ), or stained with Ponceau S (lower panel). PC12 lysates (0.5% of that processed in the pull down reaction) were loaded for reference. Right and left panels are two separate experiments. Molecular weights are marked on the right. C. GST-fused Kv2.1-C1a protein or GST alone (150 pmole each) immobilized on glutathione affinity beads were incubated with 35 S-labeled synaptotagmin ( Syt ), 35 S-labeled syntaxin ( Syx ), 35 S-labeled SNAP-25 or 35 S-labeled VAMP2 for 1 h. in 1 ml 0.1% Triton X-100. Gluthation-eluted proteins ( right panel ) and input (20 µl out of 1000 µl of reaction mixture taken before the addition of beads; left panel ) were analyzed by SDS-PAGE.
    Figure Legend Snippet: A. A schematic presentation of the Kv2.1 channel showing fragments generated as GST fusion proteins. The grey box on the left, the 6 dark boxes and the grey box on the right denote the N terminus, the 6 trans membrane and the C terminus domains, respectively. Numbers denote amino acids. B. GST fusion proteins corresponding to Kv2.1 C terminus but not N terminus pull down syntaxin from PC12 cell lysate. Proteins (each at 150 pmol) immobilized on glutathione affinity beads were incubated with 10 6 cells in K-Glu buffer with 1% Triton X-100 for 12 hr at 4°C. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) and immunobloted with syntaxin antibody ( IB Syx ), or stained with Ponceau S (lower panel). PC12 lysates (0.5% of that processed in the pull down reaction) were loaded for reference. Right and left panels are two separate experiments. Molecular weights are marked on the right. C. GST-fused Kv2.1-C1a protein or GST alone (150 pmole each) immobilized on glutathione affinity beads were incubated with 35 S-labeled synaptotagmin ( Syt ), 35 S-labeled syntaxin ( Syx ), 35 S-labeled SNAP-25 or 35 S-labeled VAMP2 for 1 h. in 1 ml 0.1% Triton X-100. Gluthation-eluted proteins ( right panel ) and input (20 µl out of 1000 µl of reaction mixture taken before the addition of beads; left panel ) were analyzed by SDS-PAGE.

    Techniques Used: Generated, Incubation, SDS Page, Staining, Labeling

    (A) PC12 cells preloaded with [ 3 H]-NE and gently homogenized ( “cracked” cells), either underwent 15 min incubation in the presence of 2 mM MgATP and brain cytosol at 30°C (“priming”) or not, followed by either 15 min incubation with added 1.6 mM Ca 2+ (1 µM free Ca 2+ ; at 30° (“triggering”) or not, as indicated below the bars. Cells were pelleted and the secreted [ 3 H]-NE in the supernatant was quantified by scintillation counting and expressed as a percentage of the total [ 3 H]-NE in the cell. 10 6 cells per reaction were used. The difference between the amounts of [ 3 H]-NE secreted in the two reactions on the two bars on the right was defined and is referred to hereafter as the Ca 2+ -triggered release. (B) Cracked cells preloaded with [ 3 H]-NE underwent priming and triggering reactions in the absence or presence of 10–20 µM GST-fusion peptides corresponding to cytosolic parts of Kv2.1 and Kv1.1 (shown in ) or to GST itself (as indicated below bars). Values of Ca 2+ -triggered release measured in the presence of the peptides (see ) were normalized to the control release determined in the absence of peptides (defined as 100%). Each bar in A and B depicts the mean±s.e.m from several independent experiments (numbers of experiments are in parentheses above bars). **, p <0.001 (compared with GST). (C) Cells were stimulated to release NE in the presence of increasing concentrations of GST-fused Kv2.1-C1 protein. Each point in the curve represents mean±s.e.m values from several independent experiments (numbers of experiments in parentheses above bars). **, p<0.001 (compared with 10 nM GST-C1).
    Figure Legend Snippet: (A) PC12 cells preloaded with [ 3 H]-NE and gently homogenized ( “cracked” cells), either underwent 15 min incubation in the presence of 2 mM MgATP and brain cytosol at 30°C (“priming”) or not, followed by either 15 min incubation with added 1.6 mM Ca 2+ (1 µM free Ca 2+ ; at 30° (“triggering”) or not, as indicated below the bars. Cells were pelleted and the secreted [ 3 H]-NE in the supernatant was quantified by scintillation counting and expressed as a percentage of the total [ 3 H]-NE in the cell. 10 6 cells per reaction were used. The difference between the amounts of [ 3 H]-NE secreted in the two reactions on the two bars on the right was defined and is referred to hereafter as the Ca 2+ -triggered release. (B) Cracked cells preloaded with [ 3 H]-NE underwent priming and triggering reactions in the absence or presence of 10–20 µM GST-fusion peptides corresponding to cytosolic parts of Kv2.1 and Kv1.1 (shown in ) or to GST itself (as indicated below bars). Values of Ca 2+ -triggered release measured in the presence of the peptides (see ) were normalized to the control release determined in the absence of peptides (defined as 100%). Each bar in A and B depicts the mean±s.e.m from several independent experiments (numbers of experiments are in parentheses above bars). **, p <0.001 (compared with GST). (C) Cells were stimulated to release NE in the presence of increasing concentrations of GST-fused Kv2.1-C1 protein. Each point in the curve represents mean±s.e.m values from several independent experiments (numbers of experiments in parentheses above bars). **, p<0.001 (compared with 10 nM GST-C1).

    Techniques Used: Incubation

    anti kv2 1 c terminus  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 94
      Buy from Supplier

    Structured Review

    Alomone Labs anti kv2 1 c terminus
    In situ interaction of <t>Kv2.1</t> with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.
    Anti Kv2 1 C Terminus, 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
    https://www.bioz.com/result/anti kv2 1 c terminus/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv2 1 c terminus - by Bioz Stars, 2023-11
    94/100 stars

    Images

    1) Product Images from "K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin"

    Article Title: K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4006-06.2007

    In situ interaction of Kv2.1 with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.
    Figure Legend Snippet: In situ interaction of Kv2.1 with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.

    Techniques Used: In Situ, Immunoprecipitation, Western Blot

    A Kv2.1 mutant with impaired syntaxin-binding capacity does not enhance release. a, Averaged current densitiy–voltage relationships in PC12 cells transfected with the mutant channel (Kv2.1 ΔC1a; n = 6), derived as in Figure 2b. Note that the current densities of the mutant channel are similar to those of the wild-type channel (compare with Fig. 2b). b, Normalized conductance (G/Gmax)–voltage relationship of Kv2.1 ΔC1a superimposed on that of Kv2.1 (from Fig. 2c). c, Physical interaction of the Kv2.1ΔC1a channel with syntaxin in Xenopus oocytes is impaired. Digitized PhosphorImager scans of SDS-PAGE analysis of [35S]Met/Cys-labeled Kv2.1, Kv2.1ΔC1a, and syntaxin proteins coprecipitated by either anti-Kv2.1 antibody (IP Kv2.1) or anti-syntaxin antibody (IP Syx) from oocytes that were injected with Kv2.1 or Kv2.1ΔC1a together with syntaxin (+Syx), or injected with syntaxin alone (Syx) are shown. Arrows indicate the relevant proteins. The scans show that the amount of syntaxin coprecipitated with mutant channel (see IP Kv2.1) is significantly smaller than that coprecipitated with the same amount wild-type channel, although expression of syntaxin in oocytes expressing the mutant channel was not smaller than that of the wild type (see IP Syx). The results shown are from one of four independent experiments. d, Kv2.1ΔC1a overexpression does not enhance neuropeptide release induced by depolarization with high external KCl concentration. Cells were transfected with the mutant (Kv2.1ΔC1a) or with empty vector (control) (n = 12 and 10, respectively). *p < 0.05.
    Figure Legend Snippet: A Kv2.1 mutant with impaired syntaxin-binding capacity does not enhance release. a, Averaged current densitiy–voltage relationships in PC12 cells transfected with the mutant channel (Kv2.1 ΔC1a; n = 6), derived as in Figure 2b. Note that the current densities of the mutant channel are similar to those of the wild-type channel (compare with Fig. 2b). b, Normalized conductance (G/Gmax)–voltage relationship of Kv2.1 ΔC1a superimposed on that of Kv2.1 (from Fig. 2c). c, Physical interaction of the Kv2.1ΔC1a channel with syntaxin in Xenopus oocytes is impaired. Digitized PhosphorImager scans of SDS-PAGE analysis of [35S]Met/Cys-labeled Kv2.1, Kv2.1ΔC1a, and syntaxin proteins coprecipitated by either anti-Kv2.1 antibody (IP Kv2.1) or anti-syntaxin antibody (IP Syx) from oocytes that were injected with Kv2.1 or Kv2.1ΔC1a together with syntaxin (+Syx), or injected with syntaxin alone (Syx) are shown. Arrows indicate the relevant proteins. The scans show that the amount of syntaxin coprecipitated with mutant channel (see IP Kv2.1) is significantly smaller than that coprecipitated with the same amount wild-type channel, although expression of syntaxin in oocytes expressing the mutant channel was not smaller than that of the wild type (see IP Syx). The results shown are from one of four independent experiments. d, Kv2.1ΔC1a overexpression does not enhance neuropeptide release induced by depolarization with high external KCl concentration. Cells were transfected with the mutant (Kv2.1ΔC1a) or with empty vector (control) (n = 12 and 10, respectively). *p < 0.05.

    Techniques Used: Mutagenesis, Binding Assay, Transfection, Derivative Assay, SDS Page, Labeling, Injection, Expressing, Over Expression, Concentration Assay, Plasmid Preparation

    Kv2.1 enhances release induced by depolarization with high external KCl concentration. The enhancement is independent on channel conductance. a, Examples of whole-cell voltage-clamped currents recorded from a control PC12 cell transfected with proANF-GFP cDNA (top) and a cell cotrasfected with proANF-GFP and Kv2.1 cDNAs (bottom). Currents were evoked by a series of steps to different potentials (noted adjacent to trace) from a holding potential of −70 mV. b, Averaged current density–voltage relationships for control and Kv2.1 cells (n = 5 and 7, respectively). c, Activation curve for Kv2.1 cells. Normalized conductance (G/Gmax)–potential relationships were fitted to a Boltzmann equation, G/Gmax = 1/(1 + exp ((Va1/2 − V)/a), and yielded a Va1/2 (half-activation voltage) of 5.8 ± 2.26 mV and an a (slope factor) of 11.8 ± 0.42. d, Time course of release induced by depolarization with high 70 mm extracellular KCl from PC12 cells cotransfected with proANF-GFP and either Kv2.1 (Kv2.1; open circles; n = 19) or empty vector (control; filled circles; n = 17). Results are the summary of eight independent experiments. e, A pore mutant channel enhances release induced by depolarization. Cells were transfected with the mutant (Kv2.1W365C/Y380T; open circles; n = 10) or with the empty vector (control; filled circles; n = 11). *p < 0.05; **p < 0.001.
    Figure Legend Snippet: Kv2.1 enhances release induced by depolarization with high external KCl concentration. The enhancement is independent on channel conductance. a, Examples of whole-cell voltage-clamped currents recorded from a control PC12 cell transfected with proANF-GFP cDNA (top) and a cell cotrasfected with proANF-GFP and Kv2.1 cDNAs (bottom). Currents were evoked by a series of steps to different potentials (noted adjacent to trace) from a holding potential of −70 mV. b, Averaged current density–voltage relationships for control and Kv2.1 cells (n = 5 and 7, respectively). c, Activation curve for Kv2.1 cells. Normalized conductance (G/Gmax)–potential relationships were fitted to a Boltzmann equation, G/Gmax = 1/(1 + exp ((Va1/2 − V)/a), and yielded a Va1/2 (half-activation voltage) of 5.8 ± 2.26 mV and an a (slope factor) of 11.8 ± 0.42. d, Time course of release induced by depolarization with high 70 mm extracellular KCl from PC12 cells cotransfected with proANF-GFP and either Kv2.1 (Kv2.1; open circles; n = 19) or empty vector (control; filled circles; n = 17). Results are the summary of eight independent experiments. e, A pore mutant channel enhances release induced by depolarization. Cells were transfected with the mutant (Kv2.1W365C/Y380T; open circles; n = 10) or with the empty vector (control; filled circles; n = 11). *p < 0.05; **p < 0.001.

    Techniques Used: Concentration Assay, Transfection, Activation Assay, Plasmid Preparation, Mutagenesis

    Kv2.1 enhancement of release does not arise from differences in membrane potential or intracellular free Ca2+. a, Membrane potential, measured from GFP-expressing cells using the whole-cell current clamp. Increased depolarizations were observed when cells were bathed in 70 mm KCl solution (KCl) compared with control solution (rest); however, membrane potentials were similar for Kv2.1- and empty vector-transfected cells (control; 15 and 11 cells per group, respectively). b, Both resting and evoked Ca2+ levels are similar in Kv2.1-transfected and control cells. Left, In a single cell, intracellular Ca2+ concentration ([Ca2+]i; filled circles) and depletion of proANF-GFP fluorescence (fluorescence; open circles) were measured during two extracellular applications of 70 mm KCl (bars). An increased rate of fluorescence depletion indicating proANF-GFP release coincides with the rise in intracellular Ca2+. Right, Bar graph showing that resting [Ca2+]i (left bars) and peak values during KCl application are similar for Kv2.1 and control cells (n = 13 and 14, respectively).
    Figure Legend Snippet: Kv2.1 enhancement of release does not arise from differences in membrane potential or intracellular free Ca2+. a, Membrane potential, measured from GFP-expressing cells using the whole-cell current clamp. Increased depolarizations were observed when cells were bathed in 70 mm KCl solution (KCl) compared with control solution (rest); however, membrane potentials were similar for Kv2.1- and empty vector-transfected cells (control; 15 and 11 cells per group, respectively). b, Both resting and evoked Ca2+ levels are similar in Kv2.1-transfected and control cells. Left, In a single cell, intracellular Ca2+ concentration ([Ca2+]i; filled circles) and depletion of proANF-GFP fluorescence (fluorescence; open circles) were measured during two extracellular applications of 70 mm KCl (bars). An increased rate of fluorescence depletion indicating proANF-GFP release coincides with the rise in intracellular Ca2+. Right, Bar graph showing that resting [Ca2+]i (left bars) and peak values during KCl application are similar for Kv2.1 and control cells (n = 13 and 14, respectively).

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Concentration Assay, Fluorescence

    The Kv2.1-C1a peptide competes with Kv2.1 for association with syntaxin to inhibit facilitation of release by Kv2.1. a, Schematic illustration of the Kv2.1 protein. Dashed segments are domains that bind syntaxin and were used in this study. b, Immunoprecipitation (IP) from PC12 cells was performed with anti-Kv2.1 antibody in the absence (control) or presence of recombinant Kv2.1 or Kv1.1 GST-fusion peptides, or the antigen (Ag) peptide for the Kv2.1 antibody, as indicated above the lanes. The immunoprecipitated Kv2.1 and the coimmunoprecipitated syntaxin (Kv2.1 and co-IPed Syx) proteins were detected by antibodies (IB), as indicated on the left side of the blots of the top panel. Molecular weights are also marked on the left. Glutathione-Sepharose beads were added to the supernatant to pulldown syntaxin (Pulled down Syx). Precipitated proteins were immunoblotted with anti-syntaxin antibody (IB Syx) or stained with ponceau S, as indicated on the left side of the bottom panel. c, f, g, Release induced by depolarization with 70 mm extracellular KCl concentration was assessed from PC12 cells (as in Fig. 2) that were transfected with empty vector (control; n = 9; c), with Kv2.1-C1a peptide (C1a; n = 15; c), with Kv2.1 alone (Kv2.1; n = 7; f) or with Kv2.1 together with Kv2.1-C1a (Kv2.1 + C1a; n = 6; f). *p < 0.05. Normalized release at 10 min after the onset of KCl application is shown in the bar diagram (g). In all cells the molar concentration of total transfected DNA was adjusted to be equal by the empty vector. d, Averaged current density at +35 mV in cells expressing Kv2.1 + C1a (n = 9) compared with those of control and Kv2.1 cells (the latter taken from Fig. 2b). e, The normalized conductance–voltage relationship from cells expressing Kv2.1 + C1a is superimposed on that of Kv2.1 (the latter taken from Fig. 2c).
    Figure Legend Snippet: The Kv2.1-C1a peptide competes with Kv2.1 for association with syntaxin to inhibit facilitation of release by Kv2.1. a, Schematic illustration of the Kv2.1 protein. Dashed segments are domains that bind syntaxin and were used in this study. b, Immunoprecipitation (IP) from PC12 cells was performed with anti-Kv2.1 antibody in the absence (control) or presence of recombinant Kv2.1 or Kv1.1 GST-fusion peptides, or the antigen (Ag) peptide for the Kv2.1 antibody, as indicated above the lanes. The immunoprecipitated Kv2.1 and the coimmunoprecipitated syntaxin (Kv2.1 and co-IPed Syx) proteins were detected by antibodies (IB), as indicated on the left side of the blots of the top panel. Molecular weights are also marked on the left. Glutathione-Sepharose beads were added to the supernatant to pulldown syntaxin (Pulled down Syx). Precipitated proteins were immunoblotted with anti-syntaxin antibody (IB Syx) or stained with ponceau S, as indicated on the left side of the bottom panel. c, f, g, Release induced by depolarization with 70 mm extracellular KCl concentration was assessed from PC12 cells (as in Fig. 2) that were transfected with empty vector (control; n = 9; c), with Kv2.1-C1a peptide (C1a; n = 15; c), with Kv2.1 alone (Kv2.1; n = 7; f) or with Kv2.1 together with Kv2.1-C1a (Kv2.1 + C1a; n = 6; f). *p < 0.05. Normalized release at 10 min after the onset of KCl application is shown in the bar diagram (g). In all cells the molar concentration of total transfected DNA was adjusted to be equal by the empty vector. d, Averaged current density at +35 mV in cells expressing Kv2.1 + C1a (n = 9) compared with those of control and Kv2.1 cells (the latter taken from Fig. 2b). e, The normalized conductance–voltage relationship from cells expressing Kv2.1 + C1a is superimposed on that of Kv2.1 (the latter taken from Fig. 2c).

    Techniques Used: Immunoprecipitation, Recombinant, Staining, Concentration Assay, Transfection, Plasmid Preparation, Expressing

    anti kv2 1 c terminus  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 93
      Buy from Supplier

    Structured Review

    Alomone Labs anti kv2 1 c terminus
    Anti Kv2 1 C Terminus, 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
    https://www.bioz.com/result/anti kv2 1 c terminus/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv2 1 c terminus - by Bioz Stars, 2023-11
    93/100 stars

    Images

    anti kv2 1 c terminus  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 86
      Buy from Supplier

    Structured Review

    Alomone Labs anti kv2 1 c terminus
    In situ interaction of <t>Kv2.1</t> with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.
    Anti Kv2 1 C Terminus, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kv2 1 c terminus/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv2 1 c terminus - by Bioz Stars, 2023-11
    86/100 stars

    Images

    1) Product Images from "K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin"

    Article Title: K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4006-06.2007

    In situ interaction of Kv2.1 with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.
    Figure Legend Snippet: In situ interaction of Kv2.1 with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.

    Techniques Used: In Situ, Immunoprecipitation, Western Blot

    A Kv2.1 mutant with impaired syntaxin-binding capacity does not enhance release. a, Averaged current densitiy–voltage relationships in PC12 cells transfected with the mutant channel (Kv2.1 ΔC1a; n = 6), derived as in Figure 2b. Note that the current densities of the mutant channel are similar to those of the wild-type channel (compare with Fig. 2b). b, Normalized conductance (G/Gmax)–voltage relationship of Kv2.1 ΔC1a superimposed on that of Kv2.1 (from Fig. 2c). c, Physical interaction of the Kv2.1ΔC1a channel with syntaxin in Xenopus oocytes is impaired. Digitized PhosphorImager scans of SDS-PAGE analysis of [35S]Met/Cys-labeled Kv2.1, Kv2.1ΔC1a, and syntaxin proteins coprecipitated by either anti-Kv2.1 antibody (IP Kv2.1) or anti-syntaxin antibody (IP Syx) from oocytes that were injected with Kv2.1 or Kv2.1ΔC1a together with syntaxin (+Syx), or injected with syntaxin alone (Syx) are shown. Arrows indicate the relevant proteins. The scans show that the amount of syntaxin coprecipitated with mutant channel (see IP Kv2.1) is significantly smaller than that coprecipitated with the same amount wild-type channel, although expression of syntaxin in oocytes expressing the mutant channel was not smaller than that of the wild type (see IP Syx). The results shown are from one of four independent experiments. d, Kv2.1ΔC1a overexpression does not enhance neuropeptide release induced by depolarization with high external KCl concentration. Cells were transfected with the mutant (Kv2.1ΔC1a) or with empty vector (control) (n = 12 and 10, respectively). *p < 0.05.
    Figure Legend Snippet: A Kv2.1 mutant with impaired syntaxin-binding capacity does not enhance release. a, Averaged current densitiy–voltage relationships in PC12 cells transfected with the mutant channel (Kv2.1 ΔC1a; n = 6), derived as in Figure 2b. Note that the current densities of the mutant channel are similar to those of the wild-type channel (compare with Fig. 2b). b, Normalized conductance (G/Gmax)–voltage relationship of Kv2.1 ΔC1a superimposed on that of Kv2.1 (from Fig. 2c). c, Physical interaction of the Kv2.1ΔC1a channel with syntaxin in Xenopus oocytes is impaired. Digitized PhosphorImager scans of SDS-PAGE analysis of [35S]Met/Cys-labeled Kv2.1, Kv2.1ΔC1a, and syntaxin proteins coprecipitated by either anti-Kv2.1 antibody (IP Kv2.1) or anti-syntaxin antibody (IP Syx) from oocytes that were injected with Kv2.1 or Kv2.1ΔC1a together with syntaxin (+Syx), or injected with syntaxin alone (Syx) are shown. Arrows indicate the relevant proteins. The scans show that the amount of syntaxin coprecipitated with mutant channel (see IP Kv2.1) is significantly smaller than that coprecipitated with the same amount wild-type channel, although expression of syntaxin in oocytes expressing the mutant channel was not smaller than that of the wild type (see IP Syx). The results shown are from one of four independent experiments. d, Kv2.1ΔC1a overexpression does not enhance neuropeptide release induced by depolarization with high external KCl concentration. Cells were transfected with the mutant (Kv2.1ΔC1a) or with empty vector (control) (n = 12 and 10, respectively). *p < 0.05.

    Techniques Used: Mutagenesis, Binding Assay, Transfection, Derivative Assay, SDS Page, Labeling, Injection, Expressing, Over Expression, Concentration Assay, Plasmid Preparation

    Kv2.1 enhances release induced by depolarization with high external KCl concentration. The enhancement is independent on channel conductance. a, Examples of whole-cell voltage-clamped currents recorded from a control PC12 cell transfected with proANF-GFP cDNA (top) and a cell cotrasfected with proANF-GFP and Kv2.1 cDNAs (bottom). Currents were evoked by a series of steps to different potentials (noted adjacent to trace) from a holding potential of −70 mV. b, Averaged current density–voltage relationships for control and Kv2.1 cells (n = 5 and 7, respectively). c, Activation curve for Kv2.1 cells. Normalized conductance (G/Gmax)–potential relationships were fitted to a Boltzmann equation, G/Gmax = 1/(1 + exp ((Va1/2 − V)/a), and yielded a Va1/2 (half-activation voltage) of 5.8 ± 2.26 mV and an a (slope factor) of 11.8 ± 0.42. d, Time course of release induced by depolarization with high 70 mm extracellular KCl from PC12 cells cotransfected with proANF-GFP and either Kv2.1 (Kv2.1; open circles; n = 19) or empty vector (control; filled circles; n = 17). Results are the summary of eight independent experiments. e, A pore mutant channel enhances release induced by depolarization. Cells were transfected with the mutant (Kv2.1W365C/Y380T; open circles; n = 10) or with the empty vector (control; filled circles; n = 11). *p < 0.05; **p < 0.001.
    Figure Legend Snippet: Kv2.1 enhances release induced by depolarization with high external KCl concentration. The enhancement is independent on channel conductance. a, Examples of whole-cell voltage-clamped currents recorded from a control PC12 cell transfected with proANF-GFP cDNA (top) and a cell cotrasfected with proANF-GFP and Kv2.1 cDNAs (bottom). Currents were evoked by a series of steps to different potentials (noted adjacent to trace) from a holding potential of −70 mV. b, Averaged current density–voltage relationships for control and Kv2.1 cells (n = 5 and 7, respectively). c, Activation curve for Kv2.1 cells. Normalized conductance (G/Gmax)–potential relationships were fitted to a Boltzmann equation, G/Gmax = 1/(1 + exp ((Va1/2 − V)/a), and yielded a Va1/2 (half-activation voltage) of 5.8 ± 2.26 mV and an a (slope factor) of 11.8 ± 0.42. d, Time course of release induced by depolarization with high 70 mm extracellular KCl from PC12 cells cotransfected with proANF-GFP and either Kv2.1 (Kv2.1; open circles; n = 19) or empty vector (control; filled circles; n = 17). Results are the summary of eight independent experiments. e, A pore mutant channel enhances release induced by depolarization. Cells were transfected with the mutant (Kv2.1W365C/Y380T; open circles; n = 10) or with the empty vector (control; filled circles; n = 11). *p < 0.05; **p < 0.001.

    Techniques Used: Concentration Assay, Transfection, Activation Assay, Plasmid Preparation, Mutagenesis

    Kv2.1 enhancement of release does not arise from differences in membrane potential or intracellular free Ca2+. a, Membrane potential, measured from GFP-expressing cells using the whole-cell current clamp. Increased depolarizations were observed when cells were bathed in 70 mm KCl solution (KCl) compared with control solution (rest); however, membrane potentials were similar for Kv2.1- and empty vector-transfected cells (control; 15 and 11 cells per group, respectively). b, Both resting and evoked Ca2+ levels are similar in Kv2.1-transfected and control cells. Left, In a single cell, intracellular Ca2+ concentration ([Ca2+]i; filled circles) and depletion of proANF-GFP fluorescence (fluorescence; open circles) were measured during two extracellular applications of 70 mm KCl (bars). An increased rate of fluorescence depletion indicating proANF-GFP release coincides with the rise in intracellular Ca2+. Right, Bar graph showing that resting [Ca2+]i (left bars) and peak values during KCl application are similar for Kv2.1 and control cells (n = 13 and 14, respectively).
    Figure Legend Snippet: Kv2.1 enhancement of release does not arise from differences in membrane potential or intracellular free Ca2+. a, Membrane potential, measured from GFP-expressing cells using the whole-cell current clamp. Increased depolarizations were observed when cells were bathed in 70 mm KCl solution (KCl) compared with control solution (rest); however, membrane potentials were similar for Kv2.1- and empty vector-transfected cells (control; 15 and 11 cells per group, respectively). b, Both resting and evoked Ca2+ levels are similar in Kv2.1-transfected and control cells. Left, In a single cell, intracellular Ca2+ concentration ([Ca2+]i; filled circles) and depletion of proANF-GFP fluorescence (fluorescence; open circles) were measured during two extracellular applications of 70 mm KCl (bars). An increased rate of fluorescence depletion indicating proANF-GFP release coincides with the rise in intracellular Ca2+. Right, Bar graph showing that resting [Ca2+]i (left bars) and peak values during KCl application are similar for Kv2.1 and control cells (n = 13 and 14, respectively).

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Concentration Assay, Fluorescence

    The Kv2.1-C1a peptide competes with Kv2.1 for association with syntaxin to inhibit facilitation of release by Kv2.1. a, Schematic illustration of the Kv2.1 protein. Dashed segments are domains that bind syntaxin and were used in this study. b, Immunoprecipitation (IP) from PC12 cells was performed with anti-Kv2.1 antibody in the absence (control) or presence of recombinant Kv2.1 or Kv1.1 GST-fusion peptides, or the antigen (Ag) peptide for the Kv2.1 antibody, as indicated above the lanes. The immunoprecipitated Kv2.1 and the coimmunoprecipitated syntaxin (Kv2.1 and co-IPed Syx) proteins were detected by antibodies (IB), as indicated on the left side of the blots of the top panel. Molecular weights are also marked on the left. Glutathione-Sepharose beads were added to the supernatant to pulldown syntaxin (Pulled down Syx). Precipitated proteins were immunoblotted with anti-syntaxin antibody (IB Syx) or stained with ponceau S, as indicated on the left side of the bottom panel. c, f, g, Release induced by depolarization with 70 mm extracellular KCl concentration was assessed from PC12 cells (as in Fig. 2) that were transfected with empty vector (control; n = 9; c), with Kv2.1-C1a peptide (C1a; n = 15; c), with Kv2.1 alone (Kv2.1; n = 7; f) or with Kv2.1 together with Kv2.1-C1a (Kv2.1 + C1a; n = 6; f). *p < 0.05. Normalized release at 10 min after the onset of KCl application is shown in the bar diagram (g). In all cells the molar concentration of total transfected DNA was adjusted to be equal by the empty vector. d, Averaged current density at +35 mV in cells expressing Kv2.1 + C1a (n = 9) compared with those of control and Kv2.1 cells (the latter taken from Fig. 2b). e, The normalized conductance–voltage relationship from cells expressing Kv2.1 + C1a is superimposed on that of Kv2.1 (the latter taken from Fig. 2c).
    Figure Legend Snippet: The Kv2.1-C1a peptide competes with Kv2.1 for association with syntaxin to inhibit facilitation of release by Kv2.1. a, Schematic illustration of the Kv2.1 protein. Dashed segments are domains that bind syntaxin and were used in this study. b, Immunoprecipitation (IP) from PC12 cells was performed with anti-Kv2.1 antibody in the absence (control) or presence of recombinant Kv2.1 or Kv1.1 GST-fusion peptides, or the antigen (Ag) peptide for the Kv2.1 antibody, as indicated above the lanes. The immunoprecipitated Kv2.1 and the coimmunoprecipitated syntaxin (Kv2.1 and co-IPed Syx) proteins were detected by antibodies (IB), as indicated on the left side of the blots of the top panel. Molecular weights are also marked on the left. Glutathione-Sepharose beads were added to the supernatant to pulldown syntaxin (Pulled down Syx). Precipitated proteins were immunoblotted with anti-syntaxin antibody (IB Syx) or stained with ponceau S, as indicated on the left side of the bottom panel. c, f, g, Release induced by depolarization with 70 mm extracellular KCl concentration was assessed from PC12 cells (as in Fig. 2) that were transfected with empty vector (control; n = 9; c), with Kv2.1-C1a peptide (C1a; n = 15; c), with Kv2.1 alone (Kv2.1; n = 7; f) or with Kv2.1 together with Kv2.1-C1a (Kv2.1 + C1a; n = 6; f). *p < 0.05. Normalized release at 10 min after the onset of KCl application is shown in the bar diagram (g). In all cells the molar concentration of total transfected DNA was adjusted to be equal by the empty vector. d, Averaged current density at +35 mV in cells expressing Kv2.1 + C1a (n = 9) compared with those of control and Kv2.1 cells (the latter taken from Fig. 2b). e, The normalized conductance–voltage relationship from cells expressing Kv2.1 + C1a is superimposed on that of Kv2.1 (the latter taken from Fig. 2c).

    Techniques Used: Immunoprecipitation, Recombinant, Staining, Concentration Assay, Transfection, Plasmid Preparation, Expressing

    anti kv2 1 c terminus  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 86
      Buy from Supplier

    Structured Review

    Alomone Labs anti kv2 1 c terminus
    In situ interaction of <t>Kv2.1</t> with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.
    Anti Kv2 1 C Terminus, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kv2 1 c terminus/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv2 1 c terminus - by Bioz Stars, 2023-11
    86/100 stars

    Images

    1) Product Images from "K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin"

    Article Title: K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4006-06.2007

    In situ interaction of Kv2.1 with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.
    Figure Legend Snippet: In situ interaction of Kv2.1 with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.

    Techniques Used: In Situ, Immunoprecipitation, Western Blot

    A Kv2.1 mutant with impaired syntaxin-binding capacity does not enhance release. a, Averaged current densitiy–voltage relationships in PC12 cells transfected with the mutant channel (Kv2.1 ΔC1a; n = 6), derived as in Figure 2b. Note that the current densities of the mutant channel are similar to those of the wild-type channel (compare with Fig. 2b). b, Normalized conductance (G/Gmax)–voltage relationship of Kv2.1 ΔC1a superimposed on that of Kv2.1 (from Fig. 2c). c, Physical interaction of the Kv2.1ΔC1a channel with syntaxin in Xenopus oocytes is impaired. Digitized PhosphorImager scans of SDS-PAGE analysis of [35S]Met/Cys-labeled Kv2.1, Kv2.1ΔC1a, and syntaxin proteins coprecipitated by either anti-Kv2.1 antibody (IP Kv2.1) or anti-syntaxin antibody (IP Syx) from oocytes that were injected with Kv2.1 or Kv2.1ΔC1a together with syntaxin (+Syx), or injected with syntaxin alone (Syx) are shown. Arrows indicate the relevant proteins. The scans show that the amount of syntaxin coprecipitated with mutant channel (see IP Kv2.1) is significantly smaller than that coprecipitated with the same amount wild-type channel, although expression of syntaxin in oocytes expressing the mutant channel was not smaller than that of the wild type (see IP Syx). The results shown are from one of four independent experiments. d, Kv2.1ΔC1a overexpression does not enhance neuropeptide release induced by depolarization with high external KCl concentration. Cells were transfected with the mutant (Kv2.1ΔC1a) or with empty vector (control) (n = 12 and 10, respectively). *p < 0.05.
    Figure Legend Snippet: A Kv2.1 mutant with impaired syntaxin-binding capacity does not enhance release. a, Averaged current densitiy–voltage relationships in PC12 cells transfected with the mutant channel (Kv2.1 ΔC1a; n = 6), derived as in Figure 2b. Note that the current densities of the mutant channel are similar to those of the wild-type channel (compare with Fig. 2b). b, Normalized conductance (G/Gmax)–voltage relationship of Kv2.1 ΔC1a superimposed on that of Kv2.1 (from Fig. 2c). c, Physical interaction of the Kv2.1ΔC1a channel with syntaxin in Xenopus oocytes is impaired. Digitized PhosphorImager scans of SDS-PAGE analysis of [35S]Met/Cys-labeled Kv2.1, Kv2.1ΔC1a, and syntaxin proteins coprecipitated by either anti-Kv2.1 antibody (IP Kv2.1) or anti-syntaxin antibody (IP Syx) from oocytes that were injected with Kv2.1 or Kv2.1ΔC1a together with syntaxin (+Syx), or injected with syntaxin alone (Syx) are shown. Arrows indicate the relevant proteins. The scans show that the amount of syntaxin coprecipitated with mutant channel (see IP Kv2.1) is significantly smaller than that coprecipitated with the same amount wild-type channel, although expression of syntaxin in oocytes expressing the mutant channel was not smaller than that of the wild type (see IP Syx). The results shown are from one of four independent experiments. d, Kv2.1ΔC1a overexpression does not enhance neuropeptide release induced by depolarization with high external KCl concentration. Cells were transfected with the mutant (Kv2.1ΔC1a) or with empty vector (control) (n = 12 and 10, respectively). *p < 0.05.

    Techniques Used: Mutagenesis, Binding Assay, Transfection, Derivative Assay, SDS Page, Labeling, Injection, Expressing, Over Expression, Concentration Assay, Plasmid Preparation

    Kv2.1 enhances release induced by depolarization with high external KCl concentration. The enhancement is independent on channel conductance. a, Examples of whole-cell voltage-clamped currents recorded from a control PC12 cell transfected with proANF-GFP cDNA (top) and a cell cotrasfected with proANF-GFP and Kv2.1 cDNAs (bottom). Currents were evoked by a series of steps to different potentials (noted adjacent to trace) from a holding potential of −70 mV. b, Averaged current density–voltage relationships for control and Kv2.1 cells (n = 5 and 7, respectively). c, Activation curve for Kv2.1 cells. Normalized conductance (G/Gmax)–potential relationships were fitted to a Boltzmann equation, G/Gmax = 1/(1 + exp ((Va1/2 − V)/a), and yielded a Va1/2 (half-activation voltage) of 5.8 ± 2.26 mV and an a (slope factor) of 11.8 ± 0.42. d, Time course of release induced by depolarization with high 70 mm extracellular KCl from PC12 cells cotransfected with proANF-GFP and either Kv2.1 (Kv2.1; open circles; n = 19) or empty vector (control; filled circles; n = 17). Results are the summary of eight independent experiments. e, A pore mutant channel enhances release induced by depolarization. Cells were transfected with the mutant (Kv2.1W365C/Y380T; open circles; n = 10) or with the empty vector (control; filled circles; n = 11). *p < 0.05; **p < 0.001.
    Figure Legend Snippet: Kv2.1 enhances release induced by depolarization with high external KCl concentration. The enhancement is independent on channel conductance. a, Examples of whole-cell voltage-clamped currents recorded from a control PC12 cell transfected with proANF-GFP cDNA (top) and a cell cotrasfected with proANF-GFP and Kv2.1 cDNAs (bottom). Currents were evoked by a series of steps to different potentials (noted adjacent to trace) from a holding potential of −70 mV. b, Averaged current density–voltage relationships for control and Kv2.1 cells (n = 5 and 7, respectively). c, Activation curve for Kv2.1 cells. Normalized conductance (G/Gmax)–potential relationships were fitted to a Boltzmann equation, G/Gmax = 1/(1 + exp ((Va1/2 − V)/a), and yielded a Va1/2 (half-activation voltage) of 5.8 ± 2.26 mV and an a (slope factor) of 11.8 ± 0.42. d, Time course of release induced by depolarization with high 70 mm extracellular KCl from PC12 cells cotransfected with proANF-GFP and either Kv2.1 (Kv2.1; open circles; n = 19) or empty vector (control; filled circles; n = 17). Results are the summary of eight independent experiments. e, A pore mutant channel enhances release induced by depolarization. Cells were transfected with the mutant (Kv2.1W365C/Y380T; open circles; n = 10) or with the empty vector (control; filled circles; n = 11). *p < 0.05; **p < 0.001.

    Techniques Used: Concentration Assay, Transfection, Activation Assay, Plasmid Preparation, Mutagenesis

    Kv2.1 enhancement of release does not arise from differences in membrane potential or intracellular free Ca2+. a, Membrane potential, measured from GFP-expressing cells using the whole-cell current clamp. Increased depolarizations were observed when cells were bathed in 70 mm KCl solution (KCl) compared with control solution (rest); however, membrane potentials were similar for Kv2.1- and empty vector-transfected cells (control; 15 and 11 cells per group, respectively). b, Both resting and evoked Ca2+ levels are similar in Kv2.1-transfected and control cells. Left, In a single cell, intracellular Ca2+ concentration ([Ca2+]i; filled circles) and depletion of proANF-GFP fluorescence (fluorescence; open circles) were measured during two extracellular applications of 70 mm KCl (bars). An increased rate of fluorescence depletion indicating proANF-GFP release coincides with the rise in intracellular Ca2+. Right, Bar graph showing that resting [Ca2+]i (left bars) and peak values during KCl application are similar for Kv2.1 and control cells (n = 13 and 14, respectively).
    Figure Legend Snippet: Kv2.1 enhancement of release does not arise from differences in membrane potential or intracellular free Ca2+. a, Membrane potential, measured from GFP-expressing cells using the whole-cell current clamp. Increased depolarizations were observed when cells were bathed in 70 mm KCl solution (KCl) compared with control solution (rest); however, membrane potentials were similar for Kv2.1- and empty vector-transfected cells (control; 15 and 11 cells per group, respectively). b, Both resting and evoked Ca2+ levels are similar in Kv2.1-transfected and control cells. Left, In a single cell, intracellular Ca2+ concentration ([Ca2+]i; filled circles) and depletion of proANF-GFP fluorescence (fluorescence; open circles) were measured during two extracellular applications of 70 mm KCl (bars). An increased rate of fluorescence depletion indicating proANF-GFP release coincides with the rise in intracellular Ca2+. Right, Bar graph showing that resting [Ca2+]i (left bars) and peak values during KCl application are similar for Kv2.1 and control cells (n = 13 and 14, respectively).

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Concentration Assay, Fluorescence

    The Kv2.1-C1a peptide competes with Kv2.1 for association with syntaxin to inhibit facilitation of release by Kv2.1. a, Schematic illustration of the Kv2.1 protein. Dashed segments are domains that bind syntaxin and were used in this study. b, Immunoprecipitation (IP) from PC12 cells was performed with anti-Kv2.1 antibody in the absence (control) or presence of recombinant Kv2.1 or Kv1.1 GST-fusion peptides, or the antigen (Ag) peptide for the Kv2.1 antibody, as indicated above the lanes. The immunoprecipitated Kv2.1 and the coimmunoprecipitated syntaxin (Kv2.1 and co-IPed Syx) proteins were detected by antibodies (IB), as indicated on the left side of the blots of the top panel. Molecular weights are also marked on the left. Glutathione-Sepharose beads were added to the supernatant to pulldown syntaxin (Pulled down Syx). Precipitated proteins were immunoblotted with anti-syntaxin antibody (IB Syx) or stained with ponceau S, as indicated on the left side of the bottom panel. c, f, g, Release induced by depolarization with 70 mm extracellular KCl concentration was assessed from PC12 cells (as in Fig. 2) that were transfected with empty vector (control; n = 9; c), with Kv2.1-C1a peptide (C1a; n = 15; c), with Kv2.1 alone (Kv2.1; n = 7; f) or with Kv2.1 together with Kv2.1-C1a (Kv2.1 + C1a; n = 6; f). *p < 0.05. Normalized release at 10 min after the onset of KCl application is shown in the bar diagram (g). In all cells the molar concentration of total transfected DNA was adjusted to be equal by the empty vector. d, Averaged current density at +35 mV in cells expressing Kv2.1 + C1a (n = 9) compared with those of control and Kv2.1 cells (the latter taken from Fig. 2b). e, The normalized conductance–voltage relationship from cells expressing Kv2.1 + C1a is superimposed on that of Kv2.1 (the latter taken from Fig. 2c).
    Figure Legend Snippet: The Kv2.1-C1a peptide competes with Kv2.1 for association with syntaxin to inhibit facilitation of release by Kv2.1. a, Schematic illustration of the Kv2.1 protein. Dashed segments are domains that bind syntaxin and were used in this study. b, Immunoprecipitation (IP) from PC12 cells was performed with anti-Kv2.1 antibody in the absence (control) or presence of recombinant Kv2.1 or Kv1.1 GST-fusion peptides, or the antigen (Ag) peptide for the Kv2.1 antibody, as indicated above the lanes. The immunoprecipitated Kv2.1 and the coimmunoprecipitated syntaxin (Kv2.1 and co-IPed Syx) proteins were detected by antibodies (IB), as indicated on the left side of the blots of the top panel. Molecular weights are also marked on the left. Glutathione-Sepharose beads were added to the supernatant to pulldown syntaxin (Pulled down Syx). Precipitated proteins were immunoblotted with anti-syntaxin antibody (IB Syx) or stained with ponceau S, as indicated on the left side of the bottom panel. c, f, g, Release induced by depolarization with 70 mm extracellular KCl concentration was assessed from PC12 cells (as in Fig. 2) that were transfected with empty vector (control; n = 9; c), with Kv2.1-C1a peptide (C1a; n = 15; c), with Kv2.1 alone (Kv2.1; n = 7; f) or with Kv2.1 together with Kv2.1-C1a (Kv2.1 + C1a; n = 6; f). *p < 0.05. Normalized release at 10 min after the onset of KCl application is shown in the bar diagram (g). In all cells the molar concentration of total transfected DNA was adjusted to be equal by the empty vector. d, Averaged current density at +35 mV in cells expressing Kv2.1 + C1a (n = 9) compared with those of control and Kv2.1 cells (the latter taken from Fig. 2b). e, The normalized conductance–voltage relationship from cells expressing Kv2.1 + C1a is superimposed on that of Kv2.1 (the latter taken from Fig. 2c).

    Techniques Used: Immunoprecipitation, Recombinant, Staining, Concentration Assay, Transfection, Plasmid Preparation, Expressing

    anti kv2 1 c terminus  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 93
      Buy from Supplier

    Structured Review

    Alomone Labs anti kv2 1 c terminus
    In situ interaction of <t>Kv2.1</t> with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.
    Anti Kv2 1 C Terminus, 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
    https://www.bioz.com/result/anti kv2 1 c terminus/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv2 1 c terminus - by Bioz Stars, 2023-11
    93/100 stars

    Images

    1) Product Images from "K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin"

    Article Title: K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4006-06.2007

    In situ interaction of Kv2.1 with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.
    Figure Legend Snippet: In situ interaction of Kv2.1 with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.

    Techniques Used: In Situ, Immunoprecipitation, Western Blot

    A Kv2.1 mutant with impaired syntaxin-binding capacity does not enhance release. a, Averaged current densitiy–voltage relationships in PC12 cells transfected with the mutant channel (Kv2.1 ΔC1a; n = 6), derived as in Figure 2b. Note that the current densities of the mutant channel are similar to those of the wild-type channel (compare with Fig. 2b). b, Normalized conductance (G/Gmax)–voltage relationship of Kv2.1 ΔC1a superimposed on that of Kv2.1 (from Fig. 2c). c, Physical interaction of the Kv2.1ΔC1a channel with syntaxin in Xenopus oocytes is impaired. Digitized PhosphorImager scans of SDS-PAGE analysis of [35S]Met/Cys-labeled Kv2.1, Kv2.1ΔC1a, and syntaxin proteins coprecipitated by either anti-Kv2.1 antibody (IP Kv2.1) or anti-syntaxin antibody (IP Syx) from oocytes that were injected with Kv2.1 or Kv2.1ΔC1a together with syntaxin (+Syx), or injected with syntaxin alone (Syx) are shown. Arrows indicate the relevant proteins. The scans show that the amount of syntaxin coprecipitated with mutant channel (see IP Kv2.1) is significantly smaller than that coprecipitated with the same amount wild-type channel, although expression of syntaxin in oocytes expressing the mutant channel was not smaller than that of the wild type (see IP Syx). The results shown are from one of four independent experiments. d, Kv2.1ΔC1a overexpression does not enhance neuropeptide release induced by depolarization with high external KCl concentration. Cells were transfected with the mutant (Kv2.1ΔC1a) or with empty vector (control) (n = 12 and 10, respectively). *p < 0.05.
    Figure Legend Snippet: A Kv2.1 mutant with impaired syntaxin-binding capacity does not enhance release. a, Averaged current densitiy–voltage relationships in PC12 cells transfected with the mutant channel (Kv2.1 ΔC1a; n = 6), derived as in Figure 2b. Note that the current densities of the mutant channel are similar to those of the wild-type channel (compare with Fig. 2b). b, Normalized conductance (G/Gmax)–voltage relationship of Kv2.1 ΔC1a superimposed on that of Kv2.1 (from Fig. 2c). c, Physical interaction of the Kv2.1ΔC1a channel with syntaxin in Xenopus oocytes is impaired. Digitized PhosphorImager scans of SDS-PAGE analysis of [35S]Met/Cys-labeled Kv2.1, Kv2.1ΔC1a, and syntaxin proteins coprecipitated by either anti-Kv2.1 antibody (IP Kv2.1) or anti-syntaxin antibody (IP Syx) from oocytes that were injected with Kv2.1 or Kv2.1ΔC1a together with syntaxin (+Syx), or injected with syntaxin alone (Syx) are shown. Arrows indicate the relevant proteins. The scans show that the amount of syntaxin coprecipitated with mutant channel (see IP Kv2.1) is significantly smaller than that coprecipitated with the same amount wild-type channel, although expression of syntaxin in oocytes expressing the mutant channel was not smaller than that of the wild type (see IP Syx). The results shown are from one of four independent experiments. d, Kv2.1ΔC1a overexpression does not enhance neuropeptide release induced by depolarization with high external KCl concentration. Cells were transfected with the mutant (Kv2.1ΔC1a) or with empty vector (control) (n = 12 and 10, respectively). *p < 0.05.

    Techniques Used: Mutagenesis, Binding Assay, Transfection, Derivative Assay, SDS Page, Labeling, Injection, Expressing, Over Expression, Concentration Assay, Plasmid Preparation

    Kv2.1 enhances release induced by depolarization with high external KCl concentration. The enhancement is independent on channel conductance. a, Examples of whole-cell voltage-clamped currents recorded from a control PC12 cell transfected with proANF-GFP cDNA (top) and a cell cotrasfected with proANF-GFP and Kv2.1 cDNAs (bottom). Currents were evoked by a series of steps to different potentials (noted adjacent to trace) from a holding potential of −70 mV. b, Averaged current density–voltage relationships for control and Kv2.1 cells (n = 5 and 7, respectively). c, Activation curve for Kv2.1 cells. Normalized conductance (G/Gmax)–potential relationships were fitted to a Boltzmann equation, G/Gmax = 1/(1 + exp ((Va1/2 − V)/a), and yielded a Va1/2 (half-activation voltage) of 5.8 ± 2.26 mV and an a (slope factor) of 11.8 ± 0.42. d, Time course of release induced by depolarization with high 70 mm extracellular KCl from PC12 cells cotransfected with proANF-GFP and either Kv2.1 (Kv2.1; open circles; n = 19) or empty vector (control; filled circles; n = 17). Results are the summary of eight independent experiments. e, A pore mutant channel enhances release induced by depolarization. Cells were transfected with the mutant (Kv2.1W365C/Y380T; open circles; n = 10) or with the empty vector (control; filled circles; n = 11). *p < 0.05; **p < 0.001.
    Figure Legend Snippet: Kv2.1 enhances release induced by depolarization with high external KCl concentration. The enhancement is independent on channel conductance. a, Examples of whole-cell voltage-clamped currents recorded from a control PC12 cell transfected with proANF-GFP cDNA (top) and a cell cotrasfected with proANF-GFP and Kv2.1 cDNAs (bottom). Currents were evoked by a series of steps to different potentials (noted adjacent to trace) from a holding potential of −70 mV. b, Averaged current density–voltage relationships for control and Kv2.1 cells (n = 5 and 7, respectively). c, Activation curve for Kv2.1 cells. Normalized conductance (G/Gmax)–potential relationships were fitted to a Boltzmann equation, G/Gmax = 1/(1 + exp ((Va1/2 − V)/a), and yielded a Va1/2 (half-activation voltage) of 5.8 ± 2.26 mV and an a (slope factor) of 11.8 ± 0.42. d, Time course of release induced by depolarization with high 70 mm extracellular KCl from PC12 cells cotransfected with proANF-GFP and either Kv2.1 (Kv2.1; open circles; n = 19) or empty vector (control; filled circles; n = 17). Results are the summary of eight independent experiments. e, A pore mutant channel enhances release induced by depolarization. Cells were transfected with the mutant (Kv2.1W365C/Y380T; open circles; n = 10) or with the empty vector (control; filled circles; n = 11). *p < 0.05; **p < 0.001.

    Techniques Used: Concentration Assay, Transfection, Activation Assay, Plasmid Preparation, Mutagenesis

    Kv2.1 enhancement of release does not arise from differences in membrane potential or intracellular free Ca2+. a, Membrane potential, measured from GFP-expressing cells using the whole-cell current clamp. Increased depolarizations were observed when cells were bathed in 70 mm KCl solution (KCl) compared with control solution (rest); however, membrane potentials were similar for Kv2.1- and empty vector-transfected cells (control; 15 and 11 cells per group, respectively). b, Both resting and evoked Ca2+ levels are similar in Kv2.1-transfected and control cells. Left, In a single cell, intracellular Ca2+ concentration ([Ca2+]i; filled circles) and depletion of proANF-GFP fluorescence (fluorescence; open circles) were measured during two extracellular applications of 70 mm KCl (bars). An increased rate of fluorescence depletion indicating proANF-GFP release coincides with the rise in intracellular Ca2+. Right, Bar graph showing that resting [Ca2+]i (left bars) and peak values during KCl application are similar for Kv2.1 and control cells (n = 13 and 14, respectively).
    Figure Legend Snippet: Kv2.1 enhancement of release does not arise from differences in membrane potential or intracellular free Ca2+. a, Membrane potential, measured from GFP-expressing cells using the whole-cell current clamp. Increased depolarizations were observed when cells were bathed in 70 mm KCl solution (KCl) compared with control solution (rest); however, membrane potentials were similar for Kv2.1- and empty vector-transfected cells (control; 15 and 11 cells per group, respectively). b, Both resting and evoked Ca2+ levels are similar in Kv2.1-transfected and control cells. Left, In a single cell, intracellular Ca2+ concentration ([Ca2+]i; filled circles) and depletion of proANF-GFP fluorescence (fluorescence; open circles) were measured during two extracellular applications of 70 mm KCl (bars). An increased rate of fluorescence depletion indicating proANF-GFP release coincides with the rise in intracellular Ca2+. Right, Bar graph showing that resting [Ca2+]i (left bars) and peak values during KCl application are similar for Kv2.1 and control cells (n = 13 and 14, respectively).

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Concentration Assay, Fluorescence

    The Kv2.1-C1a peptide competes with Kv2.1 for association with syntaxin to inhibit facilitation of release by Kv2.1. a, Schematic illustration of the Kv2.1 protein. Dashed segments are domains that bind syntaxin and were used in this study. b, Immunoprecipitation (IP) from PC12 cells was performed with anti-Kv2.1 antibody in the absence (control) or presence of recombinant Kv2.1 or Kv1.1 GST-fusion peptides, or the antigen (Ag) peptide for the Kv2.1 antibody, as indicated above the lanes. The immunoprecipitated Kv2.1 and the coimmunoprecipitated syntaxin (Kv2.1 and co-IPed Syx) proteins were detected by antibodies (IB), as indicated on the left side of the blots of the top panel. Molecular weights are also marked on the left. Glutathione-Sepharose beads were added to the supernatant to pulldown syntaxin (Pulled down Syx). Precipitated proteins were immunoblotted with anti-syntaxin antibody (IB Syx) or stained with ponceau S, as indicated on the left side of the bottom panel. c, f, g, Release induced by depolarization with 70 mm extracellular KCl concentration was assessed from PC12 cells (as in Fig. 2) that were transfected with empty vector (control; n = 9; c), with Kv2.1-C1a peptide (C1a; n = 15; c), with Kv2.1 alone (Kv2.1; n = 7; f) or with Kv2.1 together with Kv2.1-C1a (Kv2.1 + C1a; n = 6; f). *p < 0.05. Normalized release at 10 min after the onset of KCl application is shown in the bar diagram (g). In all cells the molar concentration of total transfected DNA was adjusted to be equal by the empty vector. d, Averaged current density at +35 mV in cells expressing Kv2.1 + C1a (n = 9) compared with those of control and Kv2.1 cells (the latter taken from Fig. 2b). e, The normalized conductance–voltage relationship from cells expressing Kv2.1 + C1a is superimposed on that of Kv2.1 (the latter taken from Fig. 2c).
    Figure Legend Snippet: The Kv2.1-C1a peptide competes with Kv2.1 for association with syntaxin to inhibit facilitation of release by Kv2.1. a, Schematic illustration of the Kv2.1 protein. Dashed segments are domains that bind syntaxin and were used in this study. b, Immunoprecipitation (IP) from PC12 cells was performed with anti-Kv2.1 antibody in the absence (control) or presence of recombinant Kv2.1 or Kv1.1 GST-fusion peptides, or the antigen (Ag) peptide for the Kv2.1 antibody, as indicated above the lanes. The immunoprecipitated Kv2.1 and the coimmunoprecipitated syntaxin (Kv2.1 and co-IPed Syx) proteins were detected by antibodies (IB), as indicated on the left side of the blots of the top panel. Molecular weights are also marked on the left. Glutathione-Sepharose beads were added to the supernatant to pulldown syntaxin (Pulled down Syx). Precipitated proteins were immunoblotted with anti-syntaxin antibody (IB Syx) or stained with ponceau S, as indicated on the left side of the bottom panel. c, f, g, Release induced by depolarization with 70 mm extracellular KCl concentration was assessed from PC12 cells (as in Fig. 2) that were transfected with empty vector (control; n = 9; c), with Kv2.1-C1a peptide (C1a; n = 15; c), with Kv2.1 alone (Kv2.1; n = 7; f) or with Kv2.1 together with Kv2.1-C1a (Kv2.1 + C1a; n = 6; f). *p < 0.05. Normalized release at 10 min after the onset of KCl application is shown in the bar diagram (g). In all cells the molar concentration of total transfected DNA was adjusted to be equal by the empty vector. d, Averaged current density at +35 mV in cells expressing Kv2.1 + C1a (n = 9) compared with those of control and Kv2.1 cells (the latter taken from Fig. 2b). e, The normalized conductance–voltage relationship from cells expressing Kv2.1 + C1a is superimposed on that of Kv2.1 (the latter taken from Fig. 2c).

    Techniques Used: Immunoprecipitation, Recombinant, Staining, Concentration Assay, Transfection, Plasmid Preparation, Expressing

    kv2 1 c terminus  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 86
      Buy from Supplier

    Structured Review

    Alomone Labs kv2 1 c terminus
    Kv2 1 C Terminus, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kv2 1 c terminus/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kv2 1 c terminus - by Bioz Stars, 2023-11
    86/100 stars

    Images

    kv2 1 c terminus  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 93
      Buy from Supplier

    Structured Review

    Alomone Labs kv2 1 c terminus
    Kv2 1 C Terminus, 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
    https://www.bioz.com/result/kv2 1 c terminus/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kv2 1 c terminus - by Bioz Stars, 2023-11
    93/100 stars

    Images

    kv2 1 c terminus  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 86
      Buy from Supplier

    Structured Review

    Alomone Labs kv2 1 c terminus
    Kv2 1 C Terminus, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kv2 1 c terminus/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kv2 1 c terminus - by Bioz Stars, 2023-11
    86/100 stars

    Images

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • kv2 1 c terminus  (Alomone Labs)
    93
    Alomone Labs kv2 1 c terminus
    A. A schematic presentation of the <t>Kv2.1</t> channel showing fragments generated as GST fusion proteins. The grey box on the left, the 6 dark boxes and the grey box on the right denote the N terminus, the 6 trans membrane and the C terminus domains, respectively. Numbers denote amino acids. B. GST fusion proteins corresponding to Kv2.1 C terminus but not N terminus pull down syntaxin from PC12 cell lysate. Proteins (each at 150 pmol) immobilized on glutathione affinity beads were incubated with 10 6 cells in K-Glu buffer with 1% Triton X-100 for 12 hr at 4°C. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) and immunobloted with syntaxin antibody ( IB Syx ), or stained with Ponceau S (lower panel). PC12 lysates (0.5% of that processed in the pull down reaction) were loaded for reference. Right and left panels are two separate experiments. Molecular weights are marked on the right. C. GST-fused Kv2.1-C1a protein or GST alone (150 pmole each) immobilized on glutathione affinity beads were incubated with 35 S-labeled synaptotagmin ( Syt ), 35 S-labeled syntaxin ( Syx ), 35 S-labeled SNAP-25 or 35 S-labeled VAMP2 for 1 h. in 1 ml 0.1% Triton X-100. Gluthation-eluted proteins ( right panel ) and input (20 µl out of 1000 µl of reaction mixture taken before the addition of beads; left panel ) were analyzed by SDS-PAGE.
    Kv2 1 C Terminus, 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
    https://www.bioz.com/result/kv2 1 c terminus/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kv2 1 c terminus - by Bioz Stars, 2023-11
    93/100 stars
    		  Buy from Supplier
    anti kv2 1 c terminus  (Alomone Labs)
    94
    Alomone Labs anti kv2 1 c terminus
    In situ interaction of <t>Kv2.1</t> with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.
    Anti Kv2 1 C Terminus, 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
    https://www.bioz.com/result/anti kv2 1 c terminus/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv2 1 c terminus - by Bioz Stars, 2023-11
    94/100 stars
    		  Buy from Supplier

    Image Search Results


    A. A schematic presentation of the Kv2.1 channel showing fragments generated as GST fusion proteins. The grey box on the left, the 6 dark boxes and the grey box on the right denote the N terminus, the 6 trans membrane and the C terminus domains, respectively. Numbers denote amino acids. B. GST fusion proteins corresponding to Kv2.1 C terminus but not N terminus pull down syntaxin from PC12 cell lysate. Proteins (each at 150 pmol) immobilized on glutathione affinity beads were incubated with 10 6 cells in K-Glu buffer with 1% Triton X-100 for 12 hr at 4°C. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) and immunobloted with syntaxin antibody ( IB Syx ), or stained with Ponceau S (lower panel). PC12 lysates (0.5% of that processed in the pull down reaction) were loaded for reference. Right and left panels are two separate experiments. Molecular weights are marked on the right. C. GST-fused Kv2.1-C1a protein or GST alone (150 pmole each) immobilized on glutathione affinity beads were incubated with 35 S-labeled synaptotagmin ( Syt ), 35 S-labeled syntaxin ( Syx ), 35 S-labeled SNAP-25 or 35 S-labeled VAMP2 for 1 h. in 1 ml 0.1% Triton X-100. Gluthation-eluted proteins ( right panel ) and input (20 µl out of 1000 µl of reaction mixture taken before the addition of beads; left panel ) were analyzed by SDS-PAGE.

    Journal: PLoS ONE

    Article Title: Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells

    doi: 10.1371/journal.pone.0001381

    Figure Lengend Snippet: A. A schematic presentation of the Kv2.1 channel showing fragments generated as GST fusion proteins. The grey box on the left, the 6 dark boxes and the grey box on the right denote the N terminus, the 6 trans membrane and the C terminus domains, respectively. Numbers denote amino acids. B. GST fusion proteins corresponding to Kv2.1 C terminus but not N terminus pull down syntaxin from PC12 cell lysate. Proteins (each at 150 pmol) immobilized on glutathione affinity beads were incubated with 10 6 cells in K-Glu buffer with 1% Triton X-100 for 12 hr at 4°C. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) and immunobloted with syntaxin antibody ( IB Syx ), or stained with Ponceau S (lower panel). PC12 lysates (0.5% of that processed in the pull down reaction) were loaded for reference. Right and left panels are two separate experiments. Molecular weights are marked on the right. C. GST-fused Kv2.1-C1a protein or GST alone (150 pmole each) immobilized on glutathione affinity beads were incubated with 35 S-labeled synaptotagmin ( Syt ), 35 S-labeled syntaxin ( Syx ), 35 S-labeled SNAP-25 or 35 S-labeled VAMP2 for 1 h. in 1 ml 0.1% Triton X-100. Gluthation-eluted proteins ( right panel ) and input (20 µl out of 1000 µl of reaction mixture taken before the addition of beads; left panel ) were analyzed by SDS-PAGE.

    Article Snippet: The primary antibodies used were anti Kv1.1-C terminus and Kv2.1-C terminus (Alomone Labs Jerusalem, Israel), monoclonal anti HPC-1 (Sigma Israel, Rehovot), monoclonal anti SNAP-25 (BD Transduction Lab, Lexington, KY), The DNAs of Kv1.1 and Kv2.1 fragments for production of GST fusion proteins were constructed as described previously ( , .

    Techniques: Generated, Incubation, SDS Page, Staining, Labeling

    (A) PC12 cells preloaded with [ 3 H]-NE and gently homogenized ( “cracked” cells), either underwent 15 min incubation in the presence of 2 mM MgATP and brain cytosol at 30°C (“priming”) or not, followed by either 15 min incubation with added 1.6 mM Ca 2+ (1 µM free Ca 2+ ; at 30° (“triggering”) or not, as indicated below the bars. Cells were pelleted and the secreted [ 3 H]-NE in the supernatant was quantified by scintillation counting and expressed as a percentage of the total [ 3 H]-NE in the cell. 10 6 cells per reaction were used. The difference between the amounts of [ 3 H]-NE secreted in the two reactions on the two bars on the right was defined and is referred to hereafter as the Ca 2+ -triggered release. (B) Cracked cells preloaded with [ 3 H]-NE underwent priming and triggering reactions in the absence or presence of 10–20 µM GST-fusion peptides corresponding to cytosolic parts of Kv2.1 and Kv1.1 (shown in ) or to GST itself (as indicated below bars). Values of Ca 2+ -triggered release measured in the presence of the peptides (see ) were normalized to the control release determined in the absence of peptides (defined as 100%). Each bar in A and B depicts the mean±s.e.m from several independent experiments (numbers of experiments are in parentheses above bars). **, p <0.001 (compared with GST). (C) Cells were stimulated to release NE in the presence of increasing concentrations of GST-fused Kv2.1-C1 protein. Each point in the curve represents mean±s.e.m values from several independent experiments (numbers of experiments in parentheses above bars). **, p<0.001 (compared with 10 nM GST-C1).

    Journal: PLoS ONE

    Article Title: Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells

    doi: 10.1371/journal.pone.0001381

    Figure Lengend Snippet: (A) PC12 cells preloaded with [ 3 H]-NE and gently homogenized ( “cracked” cells), either underwent 15 min incubation in the presence of 2 mM MgATP and brain cytosol at 30°C (“priming”) or not, followed by either 15 min incubation with added 1.6 mM Ca 2+ (1 µM free Ca 2+ ; at 30° (“triggering”) or not, as indicated below the bars. Cells were pelleted and the secreted [ 3 H]-NE in the supernatant was quantified by scintillation counting and expressed as a percentage of the total [ 3 H]-NE in the cell. 10 6 cells per reaction were used. The difference between the amounts of [ 3 H]-NE secreted in the two reactions on the two bars on the right was defined and is referred to hereafter as the Ca 2+ -triggered release. (B) Cracked cells preloaded with [ 3 H]-NE underwent priming and triggering reactions in the absence or presence of 10–20 µM GST-fusion peptides corresponding to cytosolic parts of Kv2.1 and Kv1.1 (shown in ) or to GST itself (as indicated below bars). Values of Ca 2+ -triggered release measured in the presence of the peptides (see ) were normalized to the control release determined in the absence of peptides (defined as 100%). Each bar in A and B depicts the mean±s.e.m from several independent experiments (numbers of experiments are in parentheses above bars). **, p <0.001 (compared with GST). (C) Cells were stimulated to release NE in the presence of increasing concentrations of GST-fused Kv2.1-C1 protein. Each point in the curve represents mean±s.e.m values from several independent experiments (numbers of experiments in parentheses above bars). **, p<0.001 (compared with 10 nM GST-C1).

    Article Snippet: The primary antibodies used were anti Kv1.1-C terminus and Kv2.1-C terminus (Alomone Labs Jerusalem, Israel), monoclonal anti HPC-1 (Sigma Israel, Rehovot), monoclonal anti SNAP-25 (BD Transduction Lab, Lexington, KY), The DNAs of Kv1.1 and Kv2.1 fragments for production of GST fusion proteins were constructed as described previously ( , .

    Techniques: Incubation

    In situ interaction of Kv2.1 with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

    doi: 10.1523/JNEUROSCI.4006-06.2007

    Figure Lengend Snippet: In situ interaction of Kv2.1 with syntaxin is dynamic. a, Syntaxin coimmunoprecipitates with Kv2.1 from permeable PC12 cells only after cross-linking of the cells with DTSSP. Permeable PC12 cells, either cross-linked (+DTSSP) or not (−DTSSP), were solubilized under stringent conditions in RIPA buffer (see Materials and Methods), immunoprecipitated by Kv2.1 or synapsin antibodies as indicated above the lanes, and detected by Western blotting with anti-syntaxin antibody. The left and right panels are two separate experiments. The experiments shown are representative of three similar experiments. b, Interaction of Kv2.1 with syntaxin is enhanced after Ca2+ triggering of primed cells. Permeable PC12 cells (3 × 106 cells) underwent priming only (primed) or priming followed by triggering (triggered) and were cross-linked with 5 mm DTSSP. Proteins were detected by Western blotting with the corresponding antibodies, as indicated on the right. Lysate (no immunoprecipitation was performed) from 1 × 105 cells was loaded (lysate). IP, Immunoprecipitated; Syx, syntaxin antibody.

    Article Snippet: The primary antibodies used were anti-Kv1.1–C terminus and anti-Kv2.1–C terminus (Alomone Labs, Jerusalem, Israel), monoclonal anti-HPC-1 (Sigma-Aldrich, Rehovot, Israel), and anti-synapsin (Calbiochem, La Jolla, CA).

    Techniques: In Situ, Immunoprecipitation, Western Blot

    A Kv2.1 mutant with impaired syntaxin-binding capacity does not enhance release. a, Averaged current densitiy–voltage relationships in PC12 cells transfected with the mutant channel (Kv2.1 ΔC1a; n = 6), derived as in Figure 2b. Note that the current densities of the mutant channel are similar to those of the wild-type channel (compare with Fig. 2b). b, Normalized conductance (G/Gmax)–voltage relationship of Kv2.1 ΔC1a superimposed on that of Kv2.1 (from Fig. 2c). c, Physical interaction of the Kv2.1ΔC1a channel with syntaxin in Xenopus oocytes is impaired. Digitized PhosphorImager scans of SDS-PAGE analysis of [35S]Met/Cys-labeled Kv2.1, Kv2.1ΔC1a, and syntaxin proteins coprecipitated by either anti-Kv2.1 antibody (IP Kv2.1) or anti-syntaxin antibody (IP Syx) from oocytes that were injected with Kv2.1 or Kv2.1ΔC1a together with syntaxin (+Syx), or injected with syntaxin alone (Syx) are shown. Arrows indicate the relevant proteins. The scans show that the amount of syntaxin coprecipitated with mutant channel (see IP Kv2.1) is significantly smaller than that coprecipitated with the same amount wild-type channel, although expression of syntaxin in oocytes expressing the mutant channel was not smaller than that of the wild type (see IP Syx). The results shown are from one of four independent experiments. d, Kv2.1ΔC1a overexpression does not enhance neuropeptide release induced by depolarization with high external KCl concentration. Cells were transfected with the mutant (Kv2.1ΔC1a) or with empty vector (control) (n = 12 and 10, respectively). *p < 0.05.

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

    doi: 10.1523/JNEUROSCI.4006-06.2007

    Figure Lengend Snippet: A Kv2.1 mutant with impaired syntaxin-binding capacity does not enhance release. a, Averaged current densitiy–voltage relationships in PC12 cells transfected with the mutant channel (Kv2.1 ΔC1a; n = 6), derived as in Figure 2b. Note that the current densities of the mutant channel are similar to those of the wild-type channel (compare with Fig. 2b). b, Normalized conductance (G/Gmax)–voltage relationship of Kv2.1 ΔC1a superimposed on that of Kv2.1 (from Fig. 2c). c, Physical interaction of the Kv2.1ΔC1a channel with syntaxin in Xenopus oocytes is impaired. Digitized PhosphorImager scans of SDS-PAGE analysis of [35S]Met/Cys-labeled Kv2.1, Kv2.1ΔC1a, and syntaxin proteins coprecipitated by either anti-Kv2.1 antibody (IP Kv2.1) or anti-syntaxin antibody (IP Syx) from oocytes that were injected with Kv2.1 or Kv2.1ΔC1a together with syntaxin (+Syx), or injected with syntaxin alone (Syx) are shown. Arrows indicate the relevant proteins. The scans show that the amount of syntaxin coprecipitated with mutant channel (see IP Kv2.1) is significantly smaller than that coprecipitated with the same amount wild-type channel, although expression of syntaxin in oocytes expressing the mutant channel was not smaller than that of the wild type (see IP Syx). The results shown are from one of four independent experiments. d, Kv2.1ΔC1a overexpression does not enhance neuropeptide release induced by depolarization with high external KCl concentration. Cells were transfected with the mutant (Kv2.1ΔC1a) or with empty vector (control) (n = 12 and 10, respectively). *p < 0.05.

    Article Snippet: The primary antibodies used were anti-Kv1.1–C terminus and anti-Kv2.1–C terminus (Alomone Labs, Jerusalem, Israel), monoclonal anti-HPC-1 (Sigma-Aldrich, Rehovot, Israel), and anti-synapsin (Calbiochem, La Jolla, CA).

    Techniques: Mutagenesis, Binding Assay, Transfection, Derivative Assay, SDS Page, Labeling, Injection, Expressing, Over Expression, Concentration Assay, Plasmid Preparation

    Kv2.1 enhances release induced by depolarization with high external KCl concentration. The enhancement is independent on channel conductance. a, Examples of whole-cell voltage-clamped currents recorded from a control PC12 cell transfected with proANF-GFP cDNA (top) and a cell cotrasfected with proANF-GFP and Kv2.1 cDNAs (bottom). Currents were evoked by a series of steps to different potentials (noted adjacent to trace) from a holding potential of −70 mV. b, Averaged current density–voltage relationships for control and Kv2.1 cells (n = 5 and 7, respectively). c, Activation curve for Kv2.1 cells. Normalized conductance (G/Gmax)–potential relationships were fitted to a Boltzmann equation, G/Gmax = 1/(1 + exp ((Va1/2 − V)/a), and yielded a Va1/2 (half-activation voltage) of 5.8 ± 2.26 mV and an a (slope factor) of 11.8 ± 0.42. d, Time course of release induced by depolarization with high 70 mm extracellular KCl from PC12 cells cotransfected with proANF-GFP and either Kv2.1 (Kv2.1; open circles; n = 19) or empty vector (control; filled circles; n = 17). Results are the summary of eight independent experiments. e, A pore mutant channel enhances release induced by depolarization. Cells were transfected with the mutant (Kv2.1W365C/Y380T; open circles; n = 10) or with the empty vector (control; filled circles; n = 11). *p < 0.05; **p < 0.001.

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

    doi: 10.1523/JNEUROSCI.4006-06.2007

    Figure Lengend Snippet: Kv2.1 enhances release induced by depolarization with high external KCl concentration. The enhancement is independent on channel conductance. a, Examples of whole-cell voltage-clamped currents recorded from a control PC12 cell transfected with proANF-GFP cDNA (top) and a cell cotrasfected with proANF-GFP and Kv2.1 cDNAs (bottom). Currents were evoked by a series of steps to different potentials (noted adjacent to trace) from a holding potential of −70 mV. b, Averaged current density–voltage relationships for control and Kv2.1 cells (n = 5 and 7, respectively). c, Activation curve for Kv2.1 cells. Normalized conductance (G/Gmax)–potential relationships were fitted to a Boltzmann equation, G/Gmax = 1/(1 + exp ((Va1/2 − V)/a), and yielded a Va1/2 (half-activation voltage) of 5.8 ± 2.26 mV and an a (slope factor) of 11.8 ± 0.42. d, Time course of release induced by depolarization with high 70 mm extracellular KCl from PC12 cells cotransfected with proANF-GFP and either Kv2.1 (Kv2.1; open circles; n = 19) or empty vector (control; filled circles; n = 17). Results are the summary of eight independent experiments. e, A pore mutant channel enhances release induced by depolarization. Cells were transfected with the mutant (Kv2.1W365C/Y380T; open circles; n = 10) or with the empty vector (control; filled circles; n = 11). *p < 0.05; **p < 0.001.

    Article Snippet: The primary antibodies used were anti-Kv1.1–C terminus and anti-Kv2.1–C terminus (Alomone Labs, Jerusalem, Israel), monoclonal anti-HPC-1 (Sigma-Aldrich, Rehovot, Israel), and anti-synapsin (Calbiochem, La Jolla, CA).

    Techniques: Concentration Assay, Transfection, Activation Assay, Plasmid Preparation, Mutagenesis

    Kv2.1 enhancement of release does not arise from differences in membrane potential or intracellular free Ca2+. a, Membrane potential, measured from GFP-expressing cells using the whole-cell current clamp. Increased depolarizations were observed when cells were bathed in 70 mm KCl solution (KCl) compared with control solution (rest); however, membrane potentials were similar for Kv2.1- and empty vector-transfected cells (control; 15 and 11 cells per group, respectively). b, Both resting and evoked Ca2+ levels are similar in Kv2.1-transfected and control cells. Left, In a single cell, intracellular Ca2+ concentration ([Ca2+]i; filled circles) and depletion of proANF-GFP fluorescence (fluorescence; open circles) were measured during two extracellular applications of 70 mm KCl (bars). An increased rate of fluorescence depletion indicating proANF-GFP release coincides with the rise in intracellular Ca2+. Right, Bar graph showing that resting [Ca2+]i (left bars) and peak values during KCl application are similar for Kv2.1 and control cells (n = 13 and 14, respectively).

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

    doi: 10.1523/JNEUROSCI.4006-06.2007

    Figure Lengend Snippet: Kv2.1 enhancement of release does not arise from differences in membrane potential or intracellular free Ca2+. a, Membrane potential, measured from GFP-expressing cells using the whole-cell current clamp. Increased depolarizations were observed when cells were bathed in 70 mm KCl solution (KCl) compared with control solution (rest); however, membrane potentials were similar for Kv2.1- and empty vector-transfected cells (control; 15 and 11 cells per group, respectively). b, Both resting and evoked Ca2+ levels are similar in Kv2.1-transfected and control cells. Left, In a single cell, intracellular Ca2+ concentration ([Ca2+]i; filled circles) and depletion of proANF-GFP fluorescence (fluorescence; open circles) were measured during two extracellular applications of 70 mm KCl (bars). An increased rate of fluorescence depletion indicating proANF-GFP release coincides with the rise in intracellular Ca2+. Right, Bar graph showing that resting [Ca2+]i (left bars) and peak values during KCl application are similar for Kv2.1 and control cells (n = 13 and 14, respectively).

    Article Snippet: The primary antibodies used were anti-Kv1.1–C terminus and anti-Kv2.1–C terminus (Alomone Labs, Jerusalem, Israel), monoclonal anti-HPC-1 (Sigma-Aldrich, Rehovot, Israel), and anti-synapsin (Calbiochem, La Jolla, CA).

    Techniques: Expressing, Plasmid Preparation, Transfection, Concentration Assay, Fluorescence

    The Kv2.1-C1a peptide competes with Kv2.1 for association with syntaxin to inhibit facilitation of release by Kv2.1. a, Schematic illustration of the Kv2.1 protein. Dashed segments are domains that bind syntaxin and were used in this study. b, Immunoprecipitation (IP) from PC12 cells was performed with anti-Kv2.1 antibody in the absence (control) or presence of recombinant Kv2.1 or Kv1.1 GST-fusion peptides, or the antigen (Ag) peptide for the Kv2.1 antibody, as indicated above the lanes. The immunoprecipitated Kv2.1 and the coimmunoprecipitated syntaxin (Kv2.1 and co-IPed Syx) proteins were detected by antibodies (IB), as indicated on the left side of the blots of the top panel. Molecular weights are also marked on the left. Glutathione-Sepharose beads were added to the supernatant to pulldown syntaxin (Pulled down Syx). Precipitated proteins were immunoblotted with anti-syntaxin antibody (IB Syx) or stained with ponceau S, as indicated on the left side of the bottom panel. c, f, g, Release induced by depolarization with 70 mm extracellular KCl concentration was assessed from PC12 cells (as in Fig. 2) that were transfected with empty vector (control; n = 9; c), with Kv2.1-C1a peptide (C1a; n = 15; c), with Kv2.1 alone (Kv2.1; n = 7; f) or with Kv2.1 together with Kv2.1-C1a (Kv2.1 + C1a; n = 6; f). *p < 0.05. Normalized release at 10 min after the onset of KCl application is shown in the bar diagram (g). In all cells the molar concentration of total transfected DNA was adjusted to be equal by the empty vector. d, Averaged current density at +35 mV in cells expressing Kv2.1 + C1a (n = 9) compared with those of control and Kv2.1 cells (the latter taken from Fig. 2b). e, The normalized conductance–voltage relationship from cells expressing Kv2.1 + C1a is superimposed on that of Kv2.1 (the latter taken from Fig. 2c).

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

    doi: 10.1523/JNEUROSCI.4006-06.2007

    Figure Lengend Snippet: The Kv2.1-C1a peptide competes with Kv2.1 for association with syntaxin to inhibit facilitation of release by Kv2.1. a, Schematic illustration of the Kv2.1 protein. Dashed segments are domains that bind syntaxin and were used in this study. b, Immunoprecipitation (IP) from PC12 cells was performed with anti-Kv2.1 antibody in the absence (control) or presence of recombinant Kv2.1 or Kv1.1 GST-fusion peptides, or the antigen (Ag) peptide for the Kv2.1 antibody, as indicated above the lanes. The immunoprecipitated Kv2.1 and the coimmunoprecipitated syntaxin (Kv2.1 and co-IPed Syx) proteins were detected by antibodies (IB), as indicated on the left side of the blots of the top panel. Molecular weights are also marked on the left. Glutathione-Sepharose beads were added to the supernatant to pulldown syntaxin (Pulled down Syx). Precipitated proteins were immunoblotted with anti-syntaxin antibody (IB Syx) or stained with ponceau S, as indicated on the left side of the bottom panel. c, f, g, Release induced by depolarization with 70 mm extracellular KCl concentration was assessed from PC12 cells (as in Fig. 2) that were transfected with empty vector (control; n = 9; c), with Kv2.1-C1a peptide (C1a; n = 15; c), with Kv2.1 alone (Kv2.1; n = 7; f) or with Kv2.1 together with Kv2.1-C1a (Kv2.1 + C1a; n = 6; f). *p < 0.05. Normalized release at 10 min after the onset of KCl application is shown in the bar diagram (g). In all cells the molar concentration of total transfected DNA was adjusted to be equal by the empty vector. d, Averaged current density at +35 mV in cells expressing Kv2.1 + C1a (n = 9) compared with those of control and Kv2.1 cells (the latter taken from Fig. 2b). e, The normalized conductance–voltage relationship from cells expressing Kv2.1 + C1a is superimposed on that of Kv2.1 (the latter taken from Fig. 2c).

    Article Snippet: The primary antibodies used were anti-Kv1.1–C terminus and anti-Kv2.1–C terminus (Alomone Labs, Jerusalem, Israel), monoclonal anti-HPC-1 (Sigma-Aldrich, Rehovot, Israel), and anti-synapsin (Calbiochem, La Jolla, CA).

    Techniques: Immunoprecipitation, Recombinant, Staining, Concentration Assay, Transfection, Plasmid Preparation, Expressing