k252a  (Alomone Labs)


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    Structured Review

    Alomone Labs k252a
    A – D , Effect of <t>K252a</t> and trkC activity on the distribution of internalized neurotrophins in retinal ganglion cells ( RGC ) ( A – C ) and SDS-PAGE analysis of internalized neurotrophins in purified RGCs ( D ). A , Labeling densities of internalized BDNF and NT-3 in the Golgi system of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. B , Labeling densities of internalized BDNF and NT-3 in lysosomes and endosomes of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. C , Quantification of anterograde transport of 50–80 ng radiolabeled NGF when coinjected in the eye with 50–60 ng cold NT-3 or BDNF. The number of experiments is indicated. Significance was determined by unpaired t test. Error bars indicate SEM. D , SDS-PAGE (15%) of internalized neurotrophins NGF, BDNF, and NT-3 recovered from purified immunopanned RGCs after 10 hr. Each sample was run with an adjacent sample of native same factor ( Na ). The molecular weight is indicated. Arrow indicates the dye front. Note that much of the BDNF recovered from RGCs is cleaved, whereas virtually all the NGF and NT-3 is intact protein by this analysis.
    K252a, 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/k252a/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    k252a - by Bioz Stars, 2022-11
    93/100 stars

    Images

    1) Product Images from "Sorting of Internalized Neurotrophins into an Endocytic Transcytosis Pathway via the Golgi System: Ultrastructural Analysis in Retinal Ganglion Cells"

    Article Title: Sorting of Internalized Neurotrophins into an Endocytic Transcytosis Pathway via the Golgi System: Ultrastructural Analysis in Retinal Ganglion Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-22-08915.2001

    A – D , Effect of K252a and trkC activity on the distribution of internalized neurotrophins in retinal ganglion cells ( RGC ) ( A – C ) and SDS-PAGE analysis of internalized neurotrophins in purified RGCs ( D ). A , Labeling densities of internalized BDNF and NT-3 in the Golgi system of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. B , Labeling densities of internalized BDNF and NT-3 in lysosomes and endosomes of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. C , Quantification of anterograde transport of 50–80 ng radiolabeled NGF when coinjected in the eye with 50–60 ng cold NT-3 or BDNF. The number of experiments is indicated. Significance was determined by unpaired t test. Error bars indicate SEM. D , SDS-PAGE (15%) of internalized neurotrophins NGF, BDNF, and NT-3 recovered from purified immunopanned RGCs after 10 hr. Each sample was run with an adjacent sample of native same factor ( Na ). The molecular weight is indicated. Arrow indicates the dye front. Note that much of the BDNF recovered from RGCs is cleaved, whereas virtually all the NGF and NT-3 is intact protein by this analysis.
    Figure Legend Snippet: A – D , Effect of K252a and trkC activity on the distribution of internalized neurotrophins in retinal ganglion cells ( RGC ) ( A – C ) and SDS-PAGE analysis of internalized neurotrophins in purified RGCs ( D ). A , Labeling densities of internalized BDNF and NT-3 in the Golgi system of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. B , Labeling densities of internalized BDNF and NT-3 in lysosomes and endosomes of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. C , Quantification of anterograde transport of 50–80 ng radiolabeled NGF when coinjected in the eye with 50–60 ng cold NT-3 or BDNF. The number of experiments is indicated. Significance was determined by unpaired t test. Error bars indicate SEM. D , SDS-PAGE (15%) of internalized neurotrophins NGF, BDNF, and NT-3 recovered from purified immunopanned RGCs after 10 hr. Each sample was run with an adjacent sample of native same factor ( Na ). The molecular weight is indicated. Arrow indicates the dye front. Note that much of the BDNF recovered from RGCs is cleaved, whereas virtually all the NGF and NT-3 is intact protein by this analysis.

    Techniques Used: Activity Assay, SDS Page, Purification, Labeling, Molecular Weight

    Internalization of 125 I-NT-3 (10–20 ng/ml) in purified retinal ganglion cells from E18–21 chick embryos does not require tyrosine kinase activity. The amount of internalization in the presence of 1 μg/ml K252a (tyrosine kinase inhibitor) is plotted as the percentage relative to the values for vehicle (1 μg/ml DMSO), which were averaged to 100% internalization (∼40,000 cpm per plate). Nonspecific association of NT-3 (at 4°C) is indicated ( dotted line ). Error bars indicate SEM. The number of independent experiments (each in duplicate) is indicated. The p level for confidence ( t test) is indicated, showing no significant effect of K252a on NT-3 internalization.
    Figure Legend Snippet: Internalization of 125 I-NT-3 (10–20 ng/ml) in purified retinal ganglion cells from E18–21 chick embryos does not require tyrosine kinase activity. The amount of internalization in the presence of 1 μg/ml K252a (tyrosine kinase inhibitor) is plotted as the percentage relative to the values for vehicle (1 μg/ml DMSO), which were averaged to 100% internalization (∼40,000 cpm per plate). Nonspecific association of NT-3 (at 4°C) is indicated ( dotted line ). Error bars indicate SEM. The number of independent experiments (each in duplicate) is indicated. The p level for confidence ( t test) is indicated, showing no significant effect of K252a on NT-3 internalization.

    Techniques Used: Purification, Activity Assay

    Diagram summarizing proposed subcellular pathways of internalized NT-3 ( black dots ) in a retinal ganglion cell. At least two pathways of internalized NT-3 can be distinguished. A lysosomal pathway of NT-3 may be common to all neurotrophins and may involve binding to the p75 receptor ( U ) or binding of NT-3 to trkC receptor ( Y ) in the presence of the tyrosine kinase inhibitor K252a. The neurotrophin is degraded in lysosomes ( LYS ). Alternatively, a novel pathway of NT-3 internalized in endosomes fuses with membranes of the Golgi apparatus: Golgi Pathway . This sorting requires tyrosine kinase activity (presumably trkC , Y ), and this pathway may join that of newly synthesized neurotrophins as well as p75 receptor ( U ) from the endoplasmic reticulum ( ER ). After passage through the Golgi system, internalized NT-3 is packaged in presumptive large dense-core vesicles ( LDCV ) for anterograde axonal transport. In this pathway, internalized NT-3 binds preferentially to p75. Anterogradely transported NT-3 is released from RGC axon terminals in the tectum, and after release it binds predominantly to trkC in tectal cells where it accumulates in multivesicular bodies ( MVB ).
    Figure Legend Snippet: Diagram summarizing proposed subcellular pathways of internalized NT-3 ( black dots ) in a retinal ganglion cell. At least two pathways of internalized NT-3 can be distinguished. A lysosomal pathway of NT-3 may be common to all neurotrophins and may involve binding to the p75 receptor ( U ) or binding of NT-3 to trkC receptor ( Y ) in the presence of the tyrosine kinase inhibitor K252a. The neurotrophin is degraded in lysosomes ( LYS ). Alternatively, a novel pathway of NT-3 internalized in endosomes fuses with membranes of the Golgi apparatus: Golgi Pathway . This sorting requires tyrosine kinase activity (presumably trkC , Y ), and this pathway may join that of newly synthesized neurotrophins as well as p75 receptor ( U ) from the endoplasmic reticulum ( ER ). After passage through the Golgi system, internalized NT-3 is packaged in presumptive large dense-core vesicles ( LDCV ) for anterograde axonal transport. In this pathway, internalized NT-3 binds preferentially to p75. Anterogradely transported NT-3 is released from RGC axon terminals in the tectum, and after release it binds predominantly to trkC in tectal cells where it accumulates in multivesicular bodies ( MVB ).

    Techniques Used: Binding Assay, Activity Assay, Synthesized

    A – C , Anterograde axonal transport of neurotrophic factors and cytochrome c in chick RGCs. A , B , The relative amount of anterograde transport of radiolabeled NT-3 was plotted by dividing the amount measured by gamma counting in the tectum (counts per minute per specific activity in picograms) by the amount measured in the eye (counts per minute per specific activity in nanograms) at the time chick embryos were killed (20 hr after injection in the eye). A , The effects of coinjection of monensin ( MON ), excess cold NT-3 ( cold NT-3 ), excess cold NGF ( cold NGF ), or excess cold BDNF ( cold BDNF ), blocking p75 antibody ( aP75 ), blocking trkC antibody ( atrkC ), and normal rabbit IgG ( IgG ). B , The effects of coinjection of monensin ( MON ), tyrosine kinase inhibitor K252a ( K252a ), vehicle ( DMSO ), wortmannin ( WOR ), LY294,002 ( LY ), and brefeldin A ( BFA ) are indicated. C , The relative amount of anterograde transport of radiolabeled glial cell line-derived neurotrophic factor ( GDNF ), cardiotrophin-1 ( CT1 ), and cytochrome c ( CytC ) was plotted by dividing the amount measured by gamma counting in the midbrain (picograms) by the amount measured in the eye (nanograms) at the time chick embryos were killed (20 hr after injection in the eye). The effects of coinjection of monensin ( MON ), excess cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM. The number of independent experiments is indicated. *** p ≤ 0.005; ** p ≤ 0.01; * p ≤ 0.05.
    Figure Legend Snippet: A – C , Anterograde axonal transport of neurotrophic factors and cytochrome c in chick RGCs. A , B , The relative amount of anterograde transport of radiolabeled NT-3 was plotted by dividing the amount measured by gamma counting in the tectum (counts per minute per specific activity in picograms) by the amount measured in the eye (counts per minute per specific activity in nanograms) at the time chick embryos were killed (20 hr after injection in the eye). A , The effects of coinjection of monensin ( MON ), excess cold NT-3 ( cold NT-3 ), excess cold NGF ( cold NGF ), or excess cold BDNF ( cold BDNF ), blocking p75 antibody ( aP75 ), blocking trkC antibody ( atrkC ), and normal rabbit IgG ( IgG ). B , The effects of coinjection of monensin ( MON ), tyrosine kinase inhibitor K252a ( K252a ), vehicle ( DMSO ), wortmannin ( WOR ), LY294,002 ( LY ), and brefeldin A ( BFA ) are indicated. C , The relative amount of anterograde transport of radiolabeled glial cell line-derived neurotrophic factor ( GDNF ), cardiotrophin-1 ( CT1 ), and cytochrome c ( CytC ) was plotted by dividing the amount measured by gamma counting in the midbrain (picograms) by the amount measured in the eye (nanograms) at the time chick embryos were killed (20 hr after injection in the eye). The effects of coinjection of monensin ( MON ), excess cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM. The number of independent experiments is indicated. *** p ≤ 0.005; ** p ≤ 0.01; * p ≤ 0.05.

    Techniques Used: Activity Assay, Injection, Blocking Assay, Derivative Assay

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    Alomone Labs anti bkα antibody
    Large-conductance Ca 2+ -activated K + channel α-subunit <t>(BKα)</t> and BKγ1 form molecular complex on the cell surface in mouse bronchial smooth muscle cells (mBSMCs). A : coimmunoprecipitation assay was performed using rat BSMs (rBSMs). Lysates were precipitated with anti-BKα antibody and blotted using either anti-BKα or anti-BKγ1 antibody. Similar results were obtained from 3 independent experiments. Two rats were used for each experiment. Lane 1 , lysates from rBSM; lane 2 , samples precipitated with control resin that cannot bind to antibodies; lane 3 , samples precipitated with resin that binds to anti-BKα antibody. B : total internal reflection fluorescence (TIRF) imaging of mBSMCs, in which BKα and BKγ1 were labeled with each antibody. Fluorescent signals from particles corresponding to BKα, BKγ1, and colocalization are shown in green, red, and yellow, respectively. Merged image was overlapped with a cell image. C : ratio of BKα particles localized alone or colocalized with BKγ1 to total BKα particles in mBSMCs (BKα alone, 134 particles and colocalized, 214 particles from 15 cells).
    Anti Bkα Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti bkα antibody/product/Alomone Labs
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    anti bkα antibody - by Bioz Stars, 2022-11
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    Large-conductance Ca 2+ -activated K + channel α-subunit (BKα) and BKγ1 form molecular complex on the cell surface in mouse bronchial smooth muscle cells (mBSMCs). A : coimmunoprecipitation assay was performed using rat BSMs (rBSMs). Lysates were precipitated with anti-BKα antibody and blotted using either anti-BKα or anti-BKγ1 antibody. Similar results were obtained from 3 independent experiments. Two rats were used for each experiment. Lane 1 , lysates from rBSM; lane 2 , samples precipitated with control resin that cannot bind to antibodies; lane 3 , samples precipitated with resin that binds to anti-BKα antibody. B : total internal reflection fluorescence (TIRF) imaging of mBSMCs, in which BKα and BKγ1 were labeled with each antibody. Fluorescent signals from particles corresponding to BKα, BKγ1, and colocalization are shown in green, red, and yellow, respectively. Merged image was overlapped with a cell image. C : ratio of BKα particles localized alone or colocalized with BKγ1 to total BKα particles in mBSMCs (BKα alone, 134 particles and colocalized, 214 particles from 15 cells).

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca2+-activated K+ channels in bronchial smooth muscle cells

    doi: 10.1152/ajplung.00331.2019

    Figure Lengend Snippet: Large-conductance Ca 2+ -activated K + channel α-subunit (BKα) and BKγ1 form molecular complex on the cell surface in mouse bronchial smooth muscle cells (mBSMCs). A : coimmunoprecipitation assay was performed using rat BSMs (rBSMs). Lysates were precipitated with anti-BKα antibody and blotted using either anti-BKα or anti-BKγ1 antibody. Similar results were obtained from 3 independent experiments. Two rats were used for each experiment. Lane 1 , lysates from rBSM; lane 2 , samples precipitated with control resin that cannot bind to antibodies; lane 3 , samples precipitated with resin that binds to anti-BKα antibody. B : total internal reflection fluorescence (TIRF) imaging of mBSMCs, in which BKα and BKγ1 were labeled with each antibody. Fluorescent signals from particles corresponding to BKα, BKγ1, and colocalization are shown in green, red, and yellow, respectively. Merged image was overlapped with a cell image. C : ratio of BKα particles localized alone or colocalized with BKγ1 to total BKα particles in mBSMCs (BKα alone, 134 particles and colocalized, 214 particles from 15 cells).

    Article Snippet: Precleared lysates (~0.5 mg of protein) were incubated with Amino Link Plus Coupling Resin (Thermo Scientific), with which 20 μg of anti-BKα antibody (APC-107; Alomone Laboratories, Jerusalem, Israel) was immobilized (overnight, 4°C).

    Techniques: Co-Immunoprecipitation Assay, Fluorescence, Imaging, Labeling

    Large-conductance Ca 2+ -activated K + channel γ1-subunit (BKγ1) expression is selectively high in mouse bronchial smooth muscle (BSM). A : real-time PCR analyses of BKγ subunits (BKγ1–4) in mouse bronchial smooth muscles (BSMs; N = 3). B : real-time PCR analyses of BKα, BKβ1, and BKγ1 in several types of mouse SM tissues ( N = 4–5). C : real-time PCR analysis was performed to compare BKγ1 mRNA expression between mouse trachea and bronchus (trachea, N = 3; bronchus, N = 5). * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca2+-activated K+ channels in bronchial smooth muscle cells

    doi: 10.1152/ajplung.00331.2019

    Figure Lengend Snippet: Large-conductance Ca 2+ -activated K + channel γ1-subunit (BKγ1) expression is selectively high in mouse bronchial smooth muscle (BSM). A : real-time PCR analyses of BKγ subunits (BKγ1–4) in mouse bronchial smooth muscles (BSMs; N = 3). B : real-time PCR analyses of BKα, BKβ1, and BKγ1 in several types of mouse SM tissues ( N = 4–5). C : real-time PCR analysis was performed to compare BKγ1 mRNA expression between mouse trachea and bronchus (trachea, N = 3; bronchus, N = 5). * P

    Article Snippet: Precleared lysates (~0.5 mg of protein) were incubated with Amino Link Plus Coupling Resin (Thermo Scientific), with which 20 μg of anti-BKα antibody (APC-107; Alomone Laboratories, Jerusalem, Israel) was immobilized (overnight, 4°C).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    BK channel protein levels are not altered by diet. A : the anti-BK α-subunit antibody detected a single protein band at ∼120 kDa consistent with previously published molecular weights. This band was eliminated by preincubating the primary

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: High-fat diet-induced obesity alters nitric oxide-mediated neuromuscular transmission and smooth muscle excitability in the mouse distal colon

    doi: 10.1152/ajpgi.00085.2016

    Figure Lengend Snippet: BK channel protein levels are not altered by diet. A : the anti-BK α-subunit antibody detected a single protein band at ∼120 kDa consistent with previously published molecular weights. This band was eliminated by preincubating the primary

    Article Snippet: Membranes were incubated for 2 h with anti-BK α-subunit primary antibody (1:20,000 dilution; APC-107; Alomone Labs, Jerusalem, Israel).

    Techniques: