k252a  (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

    Structured Review

    Alomone Labs k252a
    ApoER2 proteolysis induced by NGF depends on TrkA tyrosine kinase activity and on metalloproteinase activity. PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and (A) 100 nM <t>K252a</t> or (C) with 50 μM GM6001 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2, ApoER2-CTF and the proteolytic fragment of p75 NTR (control) were determined by western blot analysis using antibodies directed against their intracellular regions. α-tubulin is shown as a loading control, and the phosphorylated form of AKT is a control for TrkA activation by NGF. (B and D) The levels of ApoER2 CTF were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, ** P
    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, 2022-05
    93/100 stars

    Images

    1) Product Images from "Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway"

    Article Title: Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-15-108

    ApoER2 proteolysis induced by NGF depends on TrkA tyrosine kinase activity and on metalloproteinase activity. PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and (A) 100 nM K252a or (C) with 50 μM GM6001 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2, ApoER2-CTF and the proteolytic fragment of p75 NTR (control) were determined by western blot analysis using antibodies directed against their intracellular regions. α-tubulin is shown as a loading control, and the phosphorylated form of AKT is a control for TrkA activation by NGF. (B and D) The levels of ApoER2 CTF were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, ** P
    Figure Legend Snippet: ApoER2 proteolysis induced by NGF depends on TrkA tyrosine kinase activity and on metalloproteinase activity. PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and (A) 100 nM K252a or (C) with 50 μM GM6001 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2, ApoER2-CTF and the proteolytic fragment of p75 NTR (control) were determined by western blot analysis using antibodies directed against their intracellular regions. α-tubulin is shown as a loading control, and the phosphorylated form of AKT is a control for TrkA activation by NGF. (B and D) The levels of ApoER2 CTF were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, ** P

    Techniques Used: Activity Assay, Incubation, Western Blot, Activation Assay

    NGF induces the proteolytic processing of ApoER2. (A) Serum-starved PC12-ApoER2 cells were pre-treated with 10 μM DAPT (γ-secretase complex inhibitor), 50 μM GM6001 (metalloproteases inhibitor) and/or 100 nM K252a (Trk tyrosine kinase activity inhibitor) for 1 h and then incubated with 100 ng/mL NGF for 2 h. The blot shows full-length p75 NTR , p75 NTR CTF, and α-tubulin as a loading control. As described [ 40 ], NGF induced the proteolysis of p75 NTR and, thus, the accumulation of the CTF. This process depends on TrkA tyrosine kinase activity and the metalloproteinases. (B) Cells were pre-treated with 10 μM DAPT for 1 h and then incubated with 100 ng/mL NGF for 2 h. ApoER2 and the proteolytic fragment ApoER2-CTF were recognized using antibodies against the intracellular region of the receptor. α-tubulin is shown as a loading control. (C) Quantification of blot levels of ApoER2-CTF normalized to the loading control α-tubulin and plotted as the average ± SD of four independent experiments. Student’s t-test, ** P
    Figure Legend Snippet: NGF induces the proteolytic processing of ApoER2. (A) Serum-starved PC12-ApoER2 cells were pre-treated with 10 μM DAPT (γ-secretase complex inhibitor), 50 μM GM6001 (metalloproteases inhibitor) and/or 100 nM K252a (Trk tyrosine kinase activity inhibitor) for 1 h and then incubated with 100 ng/mL NGF for 2 h. The blot shows full-length p75 NTR , p75 NTR CTF, and α-tubulin as a loading control. As described [ 40 ], NGF induced the proteolysis of p75 NTR and, thus, the accumulation of the CTF. This process depends on TrkA tyrosine kinase activity and the metalloproteinases. (B) Cells were pre-treated with 10 μM DAPT for 1 h and then incubated with 100 ng/mL NGF for 2 h. ApoER2 and the proteolytic fragment ApoER2-CTF were recognized using antibodies against the intracellular region of the receptor. α-tubulin is shown as a loading control. (C) Quantification of blot levels of ApoER2-CTF normalized to the loading control α-tubulin and plotted as the average ± SD of four independent experiments. Student’s t-test, ** P

    Techniques Used: Activity Assay, Incubation

    2) 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

    3) Product Images from "Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation"

    Article Title: Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation

    Journal:

    doi: 10.1523/JNEUROSCI.2512-09.2009

    A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,
    Figure Legend Snippet: A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,

    Techniques Used: In Vitro

    4) Product Images from "Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation"

    Article Title: Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation

    Journal:

    doi: 10.1523/JNEUROSCI.2512-09.2009

    A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,
    Figure Legend Snippet: A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,

    Techniques Used: In Vitro

    5) Product Images from "BDNF–ERK–CREB signalling mediates the role of miR-132 in the regulation of the effects of oleanolic acid in male mice"

    Article Title: BDNF–ERK–CREB signalling mediates the role of miR-132 in the regulation of the effects of oleanolic acid in male mice

    Journal: Journal of Psychiatry & Neuroscience : JPN

    doi: 10.1503/jpn.130169

    Brain-derived neurotrophic factor (BDNF) is necessary for the behavioural and neurogenic effects of oleanolic acid (OA). (A) K252a suppresses the increase of sucrose preference induced by OA administration. Control versus chronic unpredictable mild stress
    Figure Legend Snippet: Brain-derived neurotrophic factor (BDNF) is necessary for the behavioural and neurogenic effects of oleanolic acid (OA). (A) K252a suppresses the increase of sucrose preference induced by OA administration. Control versus chronic unpredictable mild stress

    Techniques Used: Derivative Assay

    6) Product Images from "Regulation of neuronal excitability by release of proteins from glial cells"

    Article Title: Regulation of neuronal excitability by release of proteins from glial cells

    Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

    doi: 10.1098/rstb.2014.0194

    Blockage of NT-3 augments the upregulation of the Na + current density by thyroid hormone (T3). ( a ) Effects of incubation with 50 nM T3, 10 nM K252a and a combination of both for 4 days prior to recording. Note that co-incubation with the trk-receptor
    Figure Legend Snippet: Blockage of NT-3 augments the upregulation of the Na + current density by thyroid hormone (T3). ( a ) Effects of incubation with 50 nM T3, 10 nM K252a and a combination of both for 4 days prior to recording. Note that co-incubation with the trk-receptor

    Techniques Used: Incubation

    7) 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

    8) Product Images from "Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation"

    Article Title: Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation

    Journal:

    doi: 10.1523/JNEUROSCI.2512-09.2009

    A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,
    Figure Legend Snippet: A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,

    Techniques Used: In Vitro

    9) Product Images from "Brimonidine promotes axon growth after optic nerve injury through Erk phosphorylation"

    Article Title: Brimonidine promotes axon growth after optic nerve injury through Erk phosphorylation

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.298

    Brimonidine promotes optic nerve regeneration after the application of a Trk inhibitor. ( a ) Schematic representation of the experimental protocol used for intravitreous injection and optic nerve injury. ( b ) Western blot analyses showing the phosphorylation levels of TrkB in retinal extracts prepared from injured mice treated with brimonidine with or without a pan Trk inhibitor, K252a. Anti-TrkB antibodies were used to immunoprecipitate TrkB, and the level of TrkB phosphorylation was determined using anti-phospho-Tyr antibodies. N =3. * P
    Figure Legend Snippet: Brimonidine promotes optic nerve regeneration after the application of a Trk inhibitor. ( a ) Schematic representation of the experimental protocol used for intravitreous injection and optic nerve injury. ( b ) Western blot analyses showing the phosphorylation levels of TrkB in retinal extracts prepared from injured mice treated with brimonidine with or without a pan Trk inhibitor, K252a. Anti-TrkB antibodies were used to immunoprecipitate TrkB, and the level of TrkB phosphorylation was determined using anti-phospho-Tyr antibodies. N =3. * P

    Techniques Used: Injection, Western Blot, Mouse Assay

    10) 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

    11) Product Images from "Chronic Exposure to Nerve Growth Factor Increases Acetylcholine and Glutamate Release from Cholinergic Neurons of the Rat Medial Septum and Diagonal Band of Broca via Mechanisms Mediated by p75NTR"

    Article Title: Chronic Exposure to Nerve Growth Factor Increases Acetylcholine and Glutamate Release from Cholinergic Neurons of the Rat Medial Septum and Diagonal Band of Broca via Mechanisms Mediated by p75NTR

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4851-07.2008

    Mechanisms of NGF action. A , Example traces showing mEPSCs from cholinergic neurons grown in control (left) versus NGF condition (right). B , Kolmogorov–Smirnov graphs illustrate that NGF decreased the interevent interval (left) without altering the amplitude (right) of mEPSCs in cholinergic neurons ( n = 2 control, 3 NGF neurons). C , D , MLR2/3 (p75 NTR -antibodies), but not K252a (trk inhibitor), prevented NGF-induced increase in the amplitude of EPSCs in cholinergic neurons. n.s., p > 0.05. * p
    Figure Legend Snippet: Mechanisms of NGF action. A , Example traces showing mEPSCs from cholinergic neurons grown in control (left) versus NGF condition (right). B , Kolmogorov–Smirnov graphs illustrate that NGF decreased the interevent interval (left) without altering the amplitude (right) of mEPSCs in cholinergic neurons ( n = 2 control, 3 NGF neurons). C , D , MLR2/3 (p75 NTR -antibodies), but not K252a (trk inhibitor), prevented NGF-induced increase in the amplitude of EPSCs in cholinergic neurons. n.s., p > 0.05. * p

    Techniques Used:

    NGF increased both acetylcholine and glutamate transmission from cholinergic neurons via p75 NTR . A , Cholinergic neurons exposed to NGF displayed larger nicotinic and glutamatergic currents than controls. B , C , K252a did not affect NGF-induced increases in nicotinic or glutamatergic currents. D , E , MLR2/3 significantly reduced the effects of NGF on nicotinic and glutamatergic currents. F , G , Cholinergic neurons grown in NGF had significantly larger cell bodies compared with controls. The effect of NGF on soma size was blocked by K252a, but not by MLR2/3. n.s., p > 0.05. * p
    Figure Legend Snippet: NGF increased both acetylcholine and glutamate transmission from cholinergic neurons via p75 NTR . A , Cholinergic neurons exposed to NGF displayed larger nicotinic and glutamatergic currents than controls. B , C , K252a did not affect NGF-induced increases in nicotinic or glutamatergic currents. D , E , MLR2/3 significantly reduced the effects of NGF on nicotinic and glutamatergic currents. F , G , Cholinergic neurons grown in NGF had significantly larger cell bodies compared with controls. The effect of NGF on soma size was blocked by K252a, but not by MLR2/3. n.s., p > 0.05. * p

    Techniques Used: Transmission Assay

    12) Product Images from "Chronic Exposure to Nerve Growth Factor Increases Acetylcholine and Glutamate Release from Cholinergic Neurons of the Rat Medial Septum and Diagonal Band of Broca via Mechanisms Mediated by p75NTR"

    Article Title: Chronic Exposure to Nerve Growth Factor Increases Acetylcholine and Glutamate Release from Cholinergic Neurons of the Rat Medial Septum and Diagonal Band of Broca via Mechanisms Mediated by p75NTR

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4851-07.2008

    Mechanisms of NGF action. A , Example traces showing mEPSCs from cholinergic neurons grown in control (left) versus NGF condition (right). B , Kolmogorov–Smirnov graphs illustrate that NGF decreased the interevent interval (left) without altering the amplitude (right) of mEPSCs in cholinergic neurons ( n = 2 control, 3 NGF neurons). C , D , MLR2/3 (p75 NTR -antibodies), but not K252a (trk inhibitor), prevented NGF-induced increase in the amplitude of EPSCs in cholinergic neurons. n.s., p > 0.05. * p
    Figure Legend Snippet: Mechanisms of NGF action. A , Example traces showing mEPSCs from cholinergic neurons grown in control (left) versus NGF condition (right). B , Kolmogorov–Smirnov graphs illustrate that NGF decreased the interevent interval (left) without altering the amplitude (right) of mEPSCs in cholinergic neurons ( n = 2 control, 3 NGF neurons). C , D , MLR2/3 (p75 NTR -antibodies), but not K252a (trk inhibitor), prevented NGF-induced increase in the amplitude of EPSCs in cholinergic neurons. n.s., p > 0.05. * p

    Techniques Used:

    NGF increased both acetylcholine and glutamate transmission from cholinergic neurons via p75 NTR . A , Cholinergic neurons exposed to NGF displayed larger nicotinic and glutamatergic currents than controls. B , C , K252a did not affect NGF-induced increases in nicotinic or glutamatergic currents. D , E , MLR2/3 significantly reduced the effects of NGF on nicotinic and glutamatergic currents. F , G , Cholinergic neurons grown in NGF had significantly larger cell bodies compared with controls. The effect of NGF on soma size was blocked by K252a, but not by MLR2/3. n.s., p > 0.05. * p
    Figure Legend Snippet: NGF increased both acetylcholine and glutamate transmission from cholinergic neurons via p75 NTR . A , Cholinergic neurons exposed to NGF displayed larger nicotinic and glutamatergic currents than controls. B , C , K252a did not affect NGF-induced increases in nicotinic or glutamatergic currents. D , E , MLR2/3 significantly reduced the effects of NGF on nicotinic and glutamatergic currents. F , G , Cholinergic neurons grown in NGF had significantly larger cell bodies compared with controls. The effect of NGF on soma size was blocked by K252a, but not by MLR2/3. n.s., p > 0.05. * p

    Techniques Used: Transmission Assay

    13) Product Images from "Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice"

    Article Title: Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0988-07.2008

    Effect of the Trk receptor antagonist, K252a, on cocaine-conditioned place preference in postweanling mice. Postweanling mice were pretreated with K252a or vehicle (i.c.v.) 30 min before being conditioned with cocaine (20 mg/kg) or saline in a 7 d conditioning paradigm. On test day 8, preference scores were determined in the absence of drug administration. Postweanling mice that received cocaine only (Veh, i.c.v. plus Coc, i.p.) did not show a significant place preference for the cocaine-paired environment. However, pretreatment with K252a before cocaine (K252a, i.c.v. plus Coc, i.p.) on conditioning days 1–7 resulted in a significant place preference to the cocaine-paired environment (** p
    Figure Legend Snippet: Effect of the Trk receptor antagonist, K252a, on cocaine-conditioned place preference in postweanling mice. Postweanling mice were pretreated with K252a or vehicle (i.c.v.) 30 min before being conditioned with cocaine (20 mg/kg) or saline in a 7 d conditioning paradigm. On test day 8, preference scores were determined in the absence of drug administration. Postweanling mice that received cocaine only (Veh, i.c.v. plus Coc, i.p.) did not show a significant place preference for the cocaine-paired environment. However, pretreatment with K252a before cocaine (K252a, i.c.v. plus Coc, i.p.) on conditioning days 1–7 resulted in a significant place preference to the cocaine-paired environment (** p

    Techniques Used: Conditioned Place Preference, Mouse Assay

    Effect of the Trk receptor antagonist K252a on cocaine-induced activity in postweanling mice. Postweanling mice were pretreated daily with vehicle or K252a (i.c.v.) 30 min before saline or cocaine (20 mg/kg, i.p.) for 7 d. Activity was measured for 30 min after the saline or cocaine injections on days 1 and 7 of the study. Mice injected with cocaine had higher activity counts than those injected with saline on both days, regardless of the pretreatment (*** p
    Figure Legend Snippet: Effect of the Trk receptor antagonist K252a on cocaine-induced activity in postweanling mice. Postweanling mice were pretreated daily with vehicle or K252a (i.c.v.) 30 min before saline or cocaine (20 mg/kg, i.p.) for 7 d. Activity was measured for 30 min after the saline or cocaine injections on days 1 and 7 of the study. Mice injected with cocaine had higher activity counts than those injected with saline on both days, regardless of the pretreatment (*** p

    Techniques Used: Activity Assay, Mouse Assay, Injection

    Effects of the Trk receptor antagonist on DARPP-32 levels in the lateral caudal caudate–putamen. Postweanling mice were pretreated with K252a or vehicle intracerebroventricularly 30 min before 20 mg/kg cocaine or saline i.p. for 7 d. DARPP-32 immunoreactivity was measured in the lateral caudal caudate–putamen in tissue samples obtained 18 h after the last injection. Postweanling mice that received cocaine alone (Veh/Coc) had significantly higher levels of DARPP-32 in the lateral caudal caudate–putamen than control mice (Veh/Sal). Pretreatment with the Trk receptor antagonist K252a prevented cocaine-induced upregulation of DARPP-32 (Veh/Coc vs K252a/Coc; mean ± SEM; n = 8–10/group; * p
    Figure Legend Snippet: Effects of the Trk receptor antagonist on DARPP-32 levels in the lateral caudal caudate–putamen. Postweanling mice were pretreated with K252a or vehicle intracerebroventricularly 30 min before 20 mg/kg cocaine or saline i.p. for 7 d. DARPP-32 immunoreactivity was measured in the lateral caudal caudate–putamen in tissue samples obtained 18 h after the last injection. Postweanling mice that received cocaine alone (Veh/Coc) had significantly higher levels of DARPP-32 in the lateral caudal caudate–putamen than control mice (Veh/Sal). Pretreatment with the Trk receptor antagonist K252a prevented cocaine-induced upregulation of DARPP-32 (Veh/Coc vs K252a/Coc; mean ± SEM; n = 8–10/group; * p

    Techniques Used: Mouse Assay, Injection

    14) Product Images from "Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling"

    Article Title: Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075760

    TrkB is required for the neurite elongation by PRE-084. (A) CGNs immunostained for Sig-1R (red) and TrkB (green). Scale Bar: 10 µm. (B and C) CGNs were cultured for 24 h with or without PRE-084. As for cultures incubated with K252a, a pan-tropomyosin receptor kinase (trk) K252a was added at the same time as PRE was added to the culture. Representative images of CGNs are shown in (B). The neurite lengths were quantified from three independent experiments. The mean lengths of neurite are shown in the graph (C). Scale bar: 20 µm. (D) The same effects were observed when the cells were cultured in the presence of BDNF; n = 3, ** P
    Figure Legend Snippet: TrkB is required for the neurite elongation by PRE-084. (A) CGNs immunostained for Sig-1R (red) and TrkB (green). Scale Bar: 10 µm. (B and C) CGNs were cultured for 24 h with or without PRE-084. As for cultures incubated with K252a, a pan-tropomyosin receptor kinase (trk) K252a was added at the same time as PRE was added to the culture. Representative images of CGNs are shown in (B). The neurite lengths were quantified from three independent experiments. The mean lengths of neurite are shown in the graph (C). Scale bar: 20 µm. (D) The same effects were observed when the cells were cultured in the presence of BDNF; n = 3, ** P

    Techniques Used: Cell Culture, Incubation

    15) Product Images from "A Novel p75NTR Signaling Pathway Promotes Survival, Not Death, of Immunopurified Neocortical Subplate Neurons"

    Article Title: A Novel p75NTR Signaling Pathway Promotes Survival, Not Death, of Immunopurified Neocortical Subplate Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-14-05121.2001

    Trk signaling is not necessary for BDNF-dependent survival. a , Survival of subplate neurons grown in K252a in the absence or presence of 100 ng/ml neurotrophins. Data are normalized to survival in the absence of exogenous neurotrophin or K252a. * p
    Figure Legend Snippet: Trk signaling is not necessary for BDNF-dependent survival. a , Survival of subplate neurons grown in K252a in the absence or presence of 100 ng/ml neurotrophins. Data are normalized to survival in the absence of exogenous neurotrophin or K252a. * p

    Techniques Used:

    16) Product Images from "Regulation of neuronal excitability by release of proteins from glial cells"

    Article Title: Regulation of neuronal excitability by release of proteins from glial cells

    Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

    doi: 10.1098/rstb.2014.0194

    Blockage of NT-3 augments the upregulation of the Na + current density by thyroid hormone (T3). ( a ) Effects of incubation with 50 nM T3, 10 nM K252a and a combination of both for 4 days prior to recording. Note that co-incubation with the trk-receptor
    Figure Legend Snippet: Blockage of NT-3 augments the upregulation of the Na + current density by thyroid hormone (T3). ( a ) Effects of incubation with 50 nM T3, 10 nM K252a and a combination of both for 4 days prior to recording. Note that co-incubation with the trk-receptor

    Techniques Used: Incubation

    17) 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

    18) Product Images from "Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice"

    Article Title: Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0988-07.2008

    Effect of the Trk receptor antagonist, K252a, on cocaine-conditioned place preference in postweanling mice. Postweanling mice were pretreated with K252a or vehicle (i.c.v.) 30 min before being conditioned with cocaine (20 mg/kg) or saline in a 7 d conditioning paradigm. On test day 8, preference scores were determined in the absence of drug administration. Postweanling mice that received cocaine only (Veh, i.c.v. plus Coc, i.p.) did not show a significant place preference for the cocaine-paired environment. However, pretreatment with K252a before cocaine (K252a, i.c.v. plus Coc, i.p.) on conditioning days 1–7 resulted in a significant place preference to the cocaine-paired environment (** p
    Figure Legend Snippet: Effect of the Trk receptor antagonist, K252a, on cocaine-conditioned place preference in postweanling mice. Postweanling mice were pretreated with K252a or vehicle (i.c.v.) 30 min before being conditioned with cocaine (20 mg/kg) or saline in a 7 d conditioning paradigm. On test day 8, preference scores were determined in the absence of drug administration. Postweanling mice that received cocaine only (Veh, i.c.v. plus Coc, i.p.) did not show a significant place preference for the cocaine-paired environment. However, pretreatment with K252a before cocaine (K252a, i.c.v. plus Coc, i.p.) on conditioning days 1–7 resulted in a significant place preference to the cocaine-paired environment (** p

    Techniques Used: Conditioned Place Preference, Mouse Assay

    Effect of the Trk receptor antagonist K252a on cocaine-induced activity in postweanling mice. Postweanling mice were pretreated daily with vehicle or K252a (i.c.v.) 30 min before saline or cocaine (20 mg/kg, i.p.) for 7 d. Activity was measured for 30 min after the saline or cocaine injections on days 1 and 7 of the study. Mice injected with cocaine had higher activity counts than those injected with saline on both days, regardless of the pretreatment (*** p
    Figure Legend Snippet: Effect of the Trk receptor antagonist K252a on cocaine-induced activity in postweanling mice. Postweanling mice were pretreated daily with vehicle or K252a (i.c.v.) 30 min before saline or cocaine (20 mg/kg, i.p.) for 7 d. Activity was measured for 30 min after the saline or cocaine injections on days 1 and 7 of the study. Mice injected with cocaine had higher activity counts than those injected with saline on both days, regardless of the pretreatment (*** p

    Techniques Used: Activity Assay, Mouse Assay, Injection

    Effects of the Trk receptor antagonist on DARPP-32 levels in the lateral caudal caudate–putamen. Postweanling mice were pretreated with K252a or vehicle intracerebroventricularly 30 min before 20 mg/kg cocaine or saline i.p. for 7 d. DARPP-32 immunoreactivity was measured in the lateral caudal caudate–putamen in tissue samples obtained 18 h after the last injection. Postweanling mice that received cocaine alone (Veh/Coc) had significantly higher levels of DARPP-32 in the lateral caudal caudate–putamen than control mice (Veh/Sal). Pretreatment with the Trk receptor antagonist K252a prevented cocaine-induced upregulation of DARPP-32 (Veh/Coc vs K252a/Coc; mean ± SEM; n = 8–10/group; * p
    Figure Legend Snippet: Effects of the Trk receptor antagonist on DARPP-32 levels in the lateral caudal caudate–putamen. Postweanling mice were pretreated with K252a or vehicle intracerebroventricularly 30 min before 20 mg/kg cocaine or saline i.p. for 7 d. DARPP-32 immunoreactivity was measured in the lateral caudal caudate–putamen in tissue samples obtained 18 h after the last injection. Postweanling mice that received cocaine alone (Veh/Coc) had significantly higher levels of DARPP-32 in the lateral caudal caudate–putamen than control mice (Veh/Sal). Pretreatment with the Trk receptor antagonist K252a prevented cocaine-induced upregulation of DARPP-32 (Veh/Coc vs K252a/Coc; mean ± SEM; n = 8–10/group; * p

    Techniques Used: Mouse Assay, Injection

    19) Product Images from "A Novel p75NTR Signaling Pathway Promotes Survival, Not Death, of Immunopurified Neocortical Subplate Neurons"

    Article Title: A Novel p75NTR Signaling Pathway Promotes Survival, Not Death, of Immunopurified Neocortical Subplate Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-14-05121.2001

    Trk signaling is not necessary for BDNF-dependent survival. a , Survival of subplate neurons grown in K252a in the absence or presence of 100 ng/ml neurotrophins. Data are normalized to survival in the absence of exogenous neurotrophin or K252a. * p
    Figure Legend Snippet: Trk signaling is not necessary for BDNF-dependent survival. a , Survival of subplate neurons grown in K252a in the absence or presence of 100 ng/ml neurotrophins. Data are normalized to survival in the absence of exogenous neurotrophin or K252a. * p

    Techniques Used:

    20) Product Images from "Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice"

    Article Title: Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0988-07.2008

    Effect of the Trk receptor antagonist, K252a, on cocaine-conditioned place preference in postweanling mice. Postweanling mice were pretreated with K252a or vehicle (i.c.v.) 30 min before being conditioned with cocaine (20 mg/kg) or saline in a 7 d conditioning paradigm. On test day 8, preference scores were determined in the absence of drug administration. Postweanling mice that received cocaine only (Veh, i.c.v. plus Coc, i.p.) did not show a significant place preference for the cocaine-paired environment. However, pretreatment with K252a before cocaine (K252a, i.c.v. plus Coc, i.p.) on conditioning days 1–7 resulted in a significant place preference to the cocaine-paired environment (** p
    Figure Legend Snippet: Effect of the Trk receptor antagonist, K252a, on cocaine-conditioned place preference in postweanling mice. Postweanling mice were pretreated with K252a or vehicle (i.c.v.) 30 min before being conditioned with cocaine (20 mg/kg) or saline in a 7 d conditioning paradigm. On test day 8, preference scores were determined in the absence of drug administration. Postweanling mice that received cocaine only (Veh, i.c.v. plus Coc, i.p.) did not show a significant place preference for the cocaine-paired environment. However, pretreatment with K252a before cocaine (K252a, i.c.v. plus Coc, i.p.) on conditioning days 1–7 resulted in a significant place preference to the cocaine-paired environment (** p

    Techniques Used: Conditioned Place Preference, Mouse Assay

    Effect of the Trk receptor antagonist K252a on cocaine-induced activity in postweanling mice. Postweanling mice were pretreated daily with vehicle or K252a (i.c.v.) 30 min before saline or cocaine (20 mg/kg, i.p.) for 7 d. Activity was measured for 30 min after the saline or cocaine injections on days 1 and 7 of the study. Mice injected with cocaine had higher activity counts than those injected with saline on both days, regardless of the pretreatment (*** p
    Figure Legend Snippet: Effect of the Trk receptor antagonist K252a on cocaine-induced activity in postweanling mice. Postweanling mice were pretreated daily with vehicle or K252a (i.c.v.) 30 min before saline or cocaine (20 mg/kg, i.p.) for 7 d. Activity was measured for 30 min after the saline or cocaine injections on days 1 and 7 of the study. Mice injected with cocaine had higher activity counts than those injected with saline on both days, regardless of the pretreatment (*** p

    Techniques Used: Activity Assay, Mouse Assay, Injection

    Effects of the Trk receptor antagonist on DARPP-32 levels in the lateral caudal caudate–putamen. Postweanling mice were pretreated with K252a or vehicle intracerebroventricularly 30 min before 20 mg/kg cocaine or saline i.p. for 7 d. DARPP-32 immunoreactivity was measured in the lateral caudal caudate–putamen in tissue samples obtained 18 h after the last injection. Postweanling mice that received cocaine alone (Veh/Coc) had significantly higher levels of DARPP-32 in the lateral caudal caudate–putamen than control mice (Veh/Sal). Pretreatment with the Trk receptor antagonist K252a prevented cocaine-induced upregulation of DARPP-32 (Veh/Coc vs K252a/Coc; mean ± SEM; n = 8–10/group; * p
    Figure Legend Snippet: Effects of the Trk receptor antagonist on DARPP-32 levels in the lateral caudal caudate–putamen. Postweanling mice were pretreated with K252a or vehicle intracerebroventricularly 30 min before 20 mg/kg cocaine or saline i.p. for 7 d. DARPP-32 immunoreactivity was measured in the lateral caudal caudate–putamen in tissue samples obtained 18 h after the last injection. Postweanling mice that received cocaine alone (Veh/Coc) had significantly higher levels of DARPP-32 in the lateral caudal caudate–putamen than control mice (Veh/Sal). Pretreatment with the Trk receptor antagonist K252a prevented cocaine-induced upregulation of DARPP-32 (Veh/Coc vs K252a/Coc; mean ± SEM; n = 8–10/group; * p

    Techniques Used: Mouse Assay, Injection

    21) Product Images from "Neurotrophins are expressed in giant cell arteritis lesions and may contribute to vascular remodeling"

    Article Title: Neurotrophins are expressed in giant cell arteritis lesions and may contribute to vascular remodeling

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-014-0487-z

    Effects of NTs and the inhibitors of Trk (K252a), TrkB (ANA-12) and p75 NTR (anti-p75) on TASMC proliferation in GCA patients and controls. Proliferation assay performed with a BrdU assay on day 1 (A) in serum-starved TASMC incubated with NT or NT receptor inhibitors, or a combination of NT and NT receptor inhibitors, in GCA patients ( n = 6) and controls ( n = 10). In all figures, bars represent the mean of three experiments with their SEMs. K252a: specific Trk receptor inhibitor; ANA-12: a specific TrkB receptor inhibitor; anti-p75: p75 NTR Ab inhibitor; N (NGF) or B (BDNF) + K (K252a) or anti-p75 NTR : NGF or BDNF are added at the same time with the specific inhibitor. (B) Effect of ANA-12 on GCA-derived TASMCs proliferation. Three independent assays were performed and cells were seeded in triplicate for each cell line. BDNF, brain-derived neurotrophic factor; GCA, giant-cell arteritis; NGF, nerve growth factor; NT, neurotrophin; SEM, standard errors of the mean; TASMC, temporal artery VSMC; Trk, tropomyosin receptor kinase; VSMC, vascular smooth muscle cell.
    Figure Legend Snippet: Effects of NTs and the inhibitors of Trk (K252a), TrkB (ANA-12) and p75 NTR (anti-p75) on TASMC proliferation in GCA patients and controls. Proliferation assay performed with a BrdU assay on day 1 (A) in serum-starved TASMC incubated with NT or NT receptor inhibitors, or a combination of NT and NT receptor inhibitors, in GCA patients ( n = 6) and controls ( n = 10). In all figures, bars represent the mean of three experiments with their SEMs. K252a: specific Trk receptor inhibitor; ANA-12: a specific TrkB receptor inhibitor; anti-p75: p75 NTR Ab inhibitor; N (NGF) or B (BDNF) + K (K252a) or anti-p75 NTR : NGF or BDNF are added at the same time with the specific inhibitor. (B) Effect of ANA-12 on GCA-derived TASMCs proliferation. Three independent assays were performed and cells were seeded in triplicate for each cell line. BDNF, brain-derived neurotrophic factor; GCA, giant-cell arteritis; NGF, nerve growth factor; NT, neurotrophin; SEM, standard errors of the mean; TASMC, temporal artery VSMC; Trk, tropomyosin receptor kinase; VSMC, vascular smooth muscle cell.

    Techniques Used: Proliferation Assay, BrdU Staining, Incubation, Derivative Assay

    22) 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

    23) Product Images from "Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation"

    Article Title: Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation

    Journal:

    doi: 10.1523/JNEUROSCI.2512-09.2009

    A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,
    Figure Legend Snippet: A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,

    Techniques Used: In Vitro

    24) Product Images from "A Novel p75NTR Signaling Pathway Promotes Survival, Not Death, of Immunopurified Neocortical Subplate Neurons"

    Article Title: A Novel p75NTR Signaling Pathway Promotes Survival, Not Death, of Immunopurified Neocortical Subplate Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-14-05121.2001

    Trk signaling is not necessary for BDNF-dependent survival. a , Survival of subplate neurons grown in K252a in the absence or presence of 100 ng/ml neurotrophins. Data are normalized to survival in the absence of exogenous neurotrophin or K252a. * p
    Figure Legend Snippet: Trk signaling is not necessary for BDNF-dependent survival. a , Survival of subplate neurons grown in K252a in the absence or presence of 100 ng/ml neurotrophins. Data are normalized to survival in the absence of exogenous neurotrophin or K252a. * p

    Techniques Used:

    25) Product Images from "Regulation of neuronal excitability by release of proteins from glial cells"

    Article Title: Regulation of neuronal excitability by release of proteins from glial cells

    Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

    doi: 10.1098/rstb.2014.0194

    Blockage of NT-3 augments the upregulation of the Na + current density by thyroid hormone (T3). ( a ) Effects of incubation with 50 nM T3, 10 nM K252a and a combination of both for 4 days prior to recording. Note that co-incubation with the trk-receptor
    Figure Legend Snippet: Blockage of NT-3 augments the upregulation of the Na + current density by thyroid hormone (T3). ( a ) Effects of incubation with 50 nM T3, 10 nM K252a and a combination of both for 4 days prior to recording. Note that co-incubation with the trk-receptor

    Techniques Used: Incubation

    26) Product Images from "Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice"

    Article Title: Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0988-07.2008

    Effect of the Trk receptor antagonist, K252a, on cocaine-conditioned place preference in postweanling mice. Postweanling mice were pretreated with K252a or vehicle (i.c.v.) 30 min before being conditioned with cocaine (20 mg/kg) or saline in a 7 d conditioning paradigm. On test day 8, preference scores were determined in the absence of drug administration. Postweanling mice that received cocaine only (Veh, i.c.v. plus Coc, i.p.) did not show a significant place preference for the cocaine-paired environment. However, pretreatment with K252a before cocaine (K252a, i.c.v. plus Coc, i.p.) on conditioning days 1–7 resulted in a significant place preference to the cocaine-paired environment (** p
    Figure Legend Snippet: Effect of the Trk receptor antagonist, K252a, on cocaine-conditioned place preference in postweanling mice. Postweanling mice were pretreated with K252a or vehicle (i.c.v.) 30 min before being conditioned with cocaine (20 mg/kg) or saline in a 7 d conditioning paradigm. On test day 8, preference scores were determined in the absence of drug administration. Postweanling mice that received cocaine only (Veh, i.c.v. plus Coc, i.p.) did not show a significant place preference for the cocaine-paired environment. However, pretreatment with K252a before cocaine (K252a, i.c.v. plus Coc, i.p.) on conditioning days 1–7 resulted in a significant place preference to the cocaine-paired environment (** p

    Techniques Used: Conditioned Place Preference, Mouse Assay

    Effect of the Trk receptor antagonist K252a on cocaine-induced activity in postweanling mice. Postweanling mice were pretreated daily with vehicle or K252a (i.c.v.) 30 min before saline or cocaine (20 mg/kg, i.p.) for 7 d. Activity was measured for 30 min after the saline or cocaine injections on days 1 and 7 of the study. Mice injected with cocaine had higher activity counts than those injected with saline on both days, regardless of the pretreatment (*** p
    Figure Legend Snippet: Effect of the Trk receptor antagonist K252a on cocaine-induced activity in postweanling mice. Postweanling mice were pretreated daily with vehicle or K252a (i.c.v.) 30 min before saline or cocaine (20 mg/kg, i.p.) for 7 d. Activity was measured for 30 min after the saline or cocaine injections on days 1 and 7 of the study. Mice injected with cocaine had higher activity counts than those injected with saline on both days, regardless of the pretreatment (*** p

    Techniques Used: Activity Assay, Mouse Assay, Injection

    Effects of the Trk receptor antagonist on DARPP-32 levels in the lateral caudal caudate–putamen. Postweanling mice were pretreated with K252a or vehicle intracerebroventricularly 30 min before 20 mg/kg cocaine or saline i.p. for 7 d. DARPP-32 immunoreactivity was measured in the lateral caudal caudate–putamen in tissue samples obtained 18 h after the last injection. Postweanling mice that received cocaine alone (Veh/Coc) had significantly higher levels of DARPP-32 in the lateral caudal caudate–putamen than control mice (Veh/Sal). Pretreatment with the Trk receptor antagonist K252a prevented cocaine-induced upregulation of DARPP-32 (Veh/Coc vs K252a/Coc; mean ± SEM; n = 8–10/group; * p
    Figure Legend Snippet: Effects of the Trk receptor antagonist on DARPP-32 levels in the lateral caudal caudate–putamen. Postweanling mice were pretreated with K252a or vehicle intracerebroventricularly 30 min before 20 mg/kg cocaine or saline i.p. for 7 d. DARPP-32 immunoreactivity was measured in the lateral caudal caudate–putamen in tissue samples obtained 18 h after the last injection. Postweanling mice that received cocaine alone (Veh/Coc) had significantly higher levels of DARPP-32 in the lateral caudal caudate–putamen than control mice (Veh/Sal). Pretreatment with the Trk receptor antagonist K252a prevented cocaine-induced upregulation of DARPP-32 (Veh/Coc vs K252a/Coc; mean ± SEM; n = 8–10/group; * p

    Techniques Used: Mouse Assay, Injection

    27) Product Images from "Antidepressant-Like Effects of GM1 Ganglioside Involving the BDNF Signaling Cascade in Mice"

    Article Title: Antidepressant-Like Effects of GM1 Ganglioside Involving the BDNF Signaling Cascade in Mice

    Journal: International Journal of Neuropsychopharmacology

    doi: 10.1093/ijnp/pyw046

    K252a treatment antagonizes the effects of monosialotetrahexosylganglioside (GM1) on brain derived neurotrophic factor (BDNF) signaling cascade in the chronic social defeat stress (CSDS) model. (A) Western blot data revealed that CSDS-susceptible + GM1 + K252a mice displayed significantly lower BDNF, pTrkB, pERK1/2, pAKT, and pCREB expression in the hippocampus than CSDS-susceptible + GM1 mice (n = 6). (B) Similarly, western blot data showed that CSDS-susceptible + GM1 + K252a mice also had significantly lower BDNF, pTrkB, pERK1/2, pAKT, and pCREB expression in the medial prefrontal cortex (mPFC) than CSDS susceptible + GM1 mice (n = 6). Data are expressed as the mean ± SEM; ** P
    Figure Legend Snippet: K252a treatment antagonizes the effects of monosialotetrahexosylganglioside (GM1) on brain derived neurotrophic factor (BDNF) signaling cascade in the chronic social defeat stress (CSDS) model. (A) Western blot data revealed that CSDS-susceptible + GM1 + K252a mice displayed significantly lower BDNF, pTrkB, pERK1/2, pAKT, and pCREB expression in the hippocampus than CSDS-susceptible + GM1 mice (n = 6). (B) Similarly, western blot data showed that CSDS-susceptible + GM1 + K252a mice also had significantly lower BDNF, pTrkB, pERK1/2, pAKT, and pCREB expression in the medial prefrontal cortex (mPFC) than CSDS susceptible + GM1 mice (n = 6). Data are expressed as the mean ± SEM; ** P

    Techniques Used: Derivative Assay, Western Blot, Mouse Assay, Expressing

    Blockade of brain derived neurotrophic factor (BDNF) signaling cascade by K252a abolishes the antidepressant-like actions of monosialotetrahexosylganglioside (GM1). (A) Schematic timeline of the experimental procedure. Total 73 C57BL/6J mice were used in this experiment with 62 chronic social defeat stress (CSDS)-stressed mice and 11 nonstressed mice. CSDS-susceptible mice were co-injected with GM1 and K252a for 14 days, and behavioral tests were then performed. The vehicle refers to 0.1% DMSO in 0.9% saline. (B) The social interaction results for CSDS-susceptible mice (n = 44) and unsusceptible mice (n = 18) in this experiment. (C) Co-treatment GM1 with K252a blocked the antidepressant-like effects of GM1 in the social interaction test. CSDS-susceptible + GM1 + K252a mice displayed significantly lower social interaction than CSDS-susceptible + GM1 mice (n = 11). (D) CSDS susceptible + GM1 + K252a mice displayed significantly lower sucrose preference than CSDS susceptible + GM1 mice (n=11). (E) CSDS susceptible+GM1+K252a mice displayed significantly higher immobility time than CSDS susceptible+GM1 mice in the forced swim test (FST) (n = 11). (F) CSDS-susceptible+GM1+K252a mice also displayed significantly higher immobility time than CSDS-susceptible + GM1 mice in the tail suspension test (TST) (n = 11). Data are expressed as the mean ± SEM; ** P
    Figure Legend Snippet: Blockade of brain derived neurotrophic factor (BDNF) signaling cascade by K252a abolishes the antidepressant-like actions of monosialotetrahexosylganglioside (GM1). (A) Schematic timeline of the experimental procedure. Total 73 C57BL/6J mice were used in this experiment with 62 chronic social defeat stress (CSDS)-stressed mice and 11 nonstressed mice. CSDS-susceptible mice were co-injected with GM1 and K252a for 14 days, and behavioral tests were then performed. The vehicle refers to 0.1% DMSO in 0.9% saline. (B) The social interaction results for CSDS-susceptible mice (n = 44) and unsusceptible mice (n = 18) in this experiment. (C) Co-treatment GM1 with K252a blocked the antidepressant-like effects of GM1 in the social interaction test. CSDS-susceptible + GM1 + K252a mice displayed significantly lower social interaction than CSDS-susceptible + GM1 mice (n = 11). (D) CSDS susceptible + GM1 + K252a mice displayed significantly lower sucrose preference than CSDS susceptible + GM1 mice (n=11). (E) CSDS susceptible+GM1+K252a mice displayed significantly higher immobility time than CSDS susceptible+GM1 mice in the forced swim test (FST) (n = 11). (F) CSDS-susceptible+GM1+K252a mice also displayed significantly higher immobility time than CSDS-susceptible + GM1 mice in the tail suspension test (TST) (n = 11). Data are expressed as the mean ± SEM; ** P

    Techniques Used: Derivative Assay, Mouse Assay, Injection

    28) Product Images from "Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation"

    Article Title: Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation

    Journal:

    doi: 10.1523/JNEUROSCI.2512-09.2009

    A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,
    Figure Legend Snippet: A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,

    Techniques Used: In Vitro

    29) Product Images from "Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation"

    Article Title: Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation

    Journal:

    doi: 10.1523/JNEUROSCI.2512-09.2009

    A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,
    Figure Legend Snippet: A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,

    Techniques Used: In Vitro

    30) Product Images from "A Novel p75NTR Signaling Pathway Promotes Survival, Not Death, of Immunopurified Neocortical Subplate Neurons"

    Article Title: A Novel p75NTR Signaling Pathway Promotes Survival, Not Death, of Immunopurified Neocortical Subplate Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-14-05121.2001

    Trk signaling is not necessary for BDNF-dependent survival. a , Survival of subplate neurons grown in K252a in the absence or presence of 100 ng/ml neurotrophins. Data are normalized to survival in the absence of exogenous neurotrophin or K252a. * p
    Figure Legend Snippet: Trk signaling is not necessary for BDNF-dependent survival. a , Survival of subplate neurons grown in K252a in the absence or presence of 100 ng/ml neurotrophins. Data are normalized to survival in the absence of exogenous neurotrophin or K252a. * p

    Techniques Used:

    31) Product Images from "Regulation of neuronal excitability by release of proteins from glial cells"

    Article Title: Regulation of neuronal excitability by release of proteins from glial cells

    Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

    doi: 10.1098/rstb.2014.0194

    Blockage of NT-3 augments the upregulation of the Na + current density by thyroid hormone (T3). ( a ) Effects of incubation with 50 nM T3, 10 nM K252a and a combination of both for 4 days prior to recording. Note that co-incubation with the trk-receptor
    Figure Legend Snippet: Blockage of NT-3 augments the upregulation of the Na + current density by thyroid hormone (T3). ( a ) Effects of incubation with 50 nM T3, 10 nM K252a and a combination of both for 4 days prior to recording. Note that co-incubation with the trk-receptor

    Techniques Used: Incubation

    32) 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

    33) Product Images from "A Novel p75NTR Signaling Pathway Promotes Survival, Not Death, of Immunopurified Neocortical Subplate Neurons"

    Article Title: A Novel p75NTR Signaling Pathway Promotes Survival, Not Death, of Immunopurified Neocortical Subplate Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-14-05121.2001

    Trk signaling is not necessary for BDNF-dependent survival. a , Survival of subplate neurons grown in K252a in the absence or presence of 100 ng/ml neurotrophins. Data are normalized to survival in the absence of exogenous neurotrophin or K252a. * p
    Figure Legend Snippet: Trk signaling is not necessary for BDNF-dependent survival. a , Survival of subplate neurons grown in K252a in the absence or presence of 100 ng/ml neurotrophins. Data are normalized to survival in the absence of exogenous neurotrophin or K252a. * p

    Techniques Used:

    34) 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

    35) Product Images from "Chronic Exposure to Nerve Growth Factor Increases Acetylcholine and Glutamate Release from Cholinergic Neurons of the Rat Medial Septum and Diagonal Band of Broca via Mechanisms Mediated by p75NTR"

    Article Title: Chronic Exposure to Nerve Growth Factor Increases Acetylcholine and Glutamate Release from Cholinergic Neurons of the Rat Medial Septum and Diagonal Band of Broca via Mechanisms Mediated by p75NTR

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4851-07.2008

    Mechanisms of NGF action. A , Example traces showing mEPSCs from cholinergic neurons grown in control (left) versus NGF condition (right). B , Kolmogorov–Smirnov graphs illustrate that NGF decreased the interevent interval (left) without altering the amplitude (right) of mEPSCs in cholinergic neurons ( n = 2 control, 3 NGF neurons). C , D , MLR2/3 (p75 NTR -antibodies), but not K252a (trk inhibitor), prevented NGF-induced increase in the amplitude of EPSCs in cholinergic neurons. n.s., p > 0.05. * p
    Figure Legend Snippet: Mechanisms of NGF action. A , Example traces showing mEPSCs from cholinergic neurons grown in control (left) versus NGF condition (right). B , Kolmogorov–Smirnov graphs illustrate that NGF decreased the interevent interval (left) without altering the amplitude (right) of mEPSCs in cholinergic neurons ( n = 2 control, 3 NGF neurons). C , D , MLR2/3 (p75 NTR -antibodies), but not K252a (trk inhibitor), prevented NGF-induced increase in the amplitude of EPSCs in cholinergic neurons. n.s., p > 0.05. * p

    Techniques Used:

    NGF increased both acetylcholine and glutamate transmission from cholinergic neurons via p75 NTR . A , Cholinergic neurons exposed to NGF displayed larger nicotinic and glutamatergic currents than controls. B , C , K252a did not affect NGF-induced increases in nicotinic or glutamatergic currents. D , E , MLR2/3 significantly reduced the effects of NGF on nicotinic and glutamatergic currents. F , G , Cholinergic neurons grown in NGF had significantly larger cell bodies compared with controls. The effect of NGF on soma size was blocked by K252a, but not by MLR2/3. n.s., p > 0.05. * p
    Figure Legend Snippet: NGF increased both acetylcholine and glutamate transmission from cholinergic neurons via p75 NTR . A , Cholinergic neurons exposed to NGF displayed larger nicotinic and glutamatergic currents than controls. B , C , K252a did not affect NGF-induced increases in nicotinic or glutamatergic currents. D , E , MLR2/3 significantly reduced the effects of NGF on nicotinic and glutamatergic currents. F , G , Cholinergic neurons grown in NGF had significantly larger cell bodies compared with controls. The effect of NGF on soma size was blocked by K252a, but not by MLR2/3. n.s., p > 0.05. * p

    Techniques Used: Transmission Assay

    36) Product Images from "Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation"

    Article Title: Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation

    Journal:

    doi: 10.1523/JNEUROSCI.2512-09.2009

    A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,
    Figure Legend Snippet: A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,

    Techniques Used: In Vitro

    37) Product Images from "Chronic Exposure to Nerve Growth Factor Increases Acetylcholine and Glutamate Release from Cholinergic Neurons of the Rat Medial Septum and Diagonal Band of Broca via Mechanisms Mediated by p75NTR"

    Article Title: Chronic Exposure to Nerve Growth Factor Increases Acetylcholine and Glutamate Release from Cholinergic Neurons of the Rat Medial Septum and Diagonal Band of Broca via Mechanisms Mediated by p75NTR

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4851-07.2008

    Mechanisms of NGF action. A , Example traces showing mEPSCs from cholinergic neurons grown in control (left) versus NGF condition (right). B , Kolmogorov–Smirnov graphs illustrate that NGF decreased the interevent interval (left) without altering the amplitude (right) of mEPSCs in cholinergic neurons ( n = 2 control, 3 NGF neurons). C , D , MLR2/3 (p75 NTR -antibodies), but not K252a (trk inhibitor), prevented NGF-induced increase in the amplitude of EPSCs in cholinergic neurons. n.s., p > 0.05. * p
    Figure Legend Snippet: Mechanisms of NGF action. A , Example traces showing mEPSCs from cholinergic neurons grown in control (left) versus NGF condition (right). B , Kolmogorov–Smirnov graphs illustrate that NGF decreased the interevent interval (left) without altering the amplitude (right) of mEPSCs in cholinergic neurons ( n = 2 control, 3 NGF neurons). C , D , MLR2/3 (p75 NTR -antibodies), but not K252a (trk inhibitor), prevented NGF-induced increase in the amplitude of EPSCs in cholinergic neurons. n.s., p > 0.05. * p

    Techniques Used:

    NGF increased both acetylcholine and glutamate transmission from cholinergic neurons via p75 NTR . A , Cholinergic neurons exposed to NGF displayed larger nicotinic and glutamatergic currents than controls. B , C , K252a did not affect NGF-induced increases in nicotinic or glutamatergic currents. D , E , MLR2/3 significantly reduced the effects of NGF on nicotinic and glutamatergic currents. F , G , Cholinergic neurons grown in NGF had significantly larger cell bodies compared with controls. The effect of NGF on soma size was blocked by K252a, but not by MLR2/3. n.s., p > 0.05. * p
    Figure Legend Snippet: NGF increased both acetylcholine and glutamate transmission from cholinergic neurons via p75 NTR . A , Cholinergic neurons exposed to NGF displayed larger nicotinic and glutamatergic currents than controls. B , C , K252a did not affect NGF-induced increases in nicotinic or glutamatergic currents. D , E , MLR2/3 significantly reduced the effects of NGF on nicotinic and glutamatergic currents. F , G , Cholinergic neurons grown in NGF had significantly larger cell bodies compared with controls. The effect of NGF on soma size was blocked by K252a, but not by MLR2/3. n.s., p > 0.05. * p

    Techniques Used: Transmission Assay

    38) Product Images from "Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice"

    Article Title: Trk: A Neuromodulator of Age-Specific Behavioral and Neurochemical Responses to Cocaine in Mice

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0988-07.2008

    Effect of the Trk receptor antagonist, K252a, on cocaine-conditioned place preference in postweanling mice. Postweanling mice were pretreated with K252a or vehicle (i.c.v.) 30 min before being conditioned with cocaine (20 mg/kg) or saline in a 7 d conditioning paradigm. On test day 8, preference scores were determined in the absence of drug administration. Postweanling mice that received cocaine only (Veh, i.c.v. plus Coc, i.p.) did not show a significant place preference for the cocaine-paired environment. However, pretreatment with K252a before cocaine (K252a, i.c.v. plus Coc, i.p.) on conditioning days 1–7 resulted in a significant place preference to the cocaine-paired environment (** p
    Figure Legend Snippet: Effect of the Trk receptor antagonist, K252a, on cocaine-conditioned place preference in postweanling mice. Postweanling mice were pretreated with K252a or vehicle (i.c.v.) 30 min before being conditioned with cocaine (20 mg/kg) or saline in a 7 d conditioning paradigm. On test day 8, preference scores were determined in the absence of drug administration. Postweanling mice that received cocaine only (Veh, i.c.v. plus Coc, i.p.) did not show a significant place preference for the cocaine-paired environment. However, pretreatment with K252a before cocaine (K252a, i.c.v. plus Coc, i.p.) on conditioning days 1–7 resulted in a significant place preference to the cocaine-paired environment (** p

    Techniques Used: Conditioned Place Preference, Mouse Assay

    Effect of the Trk receptor antagonist K252a on cocaine-induced activity in postweanling mice. Postweanling mice were pretreated daily with vehicle or K252a (i.c.v.) 30 min before saline or cocaine (20 mg/kg, i.p.) for 7 d. Activity was measured for 30 min after the saline or cocaine injections on days 1 and 7 of the study. Mice injected with cocaine had higher activity counts than those injected with saline on both days, regardless of the pretreatment (*** p
    Figure Legend Snippet: Effect of the Trk receptor antagonist K252a on cocaine-induced activity in postweanling mice. Postweanling mice were pretreated daily with vehicle or K252a (i.c.v.) 30 min before saline or cocaine (20 mg/kg, i.p.) for 7 d. Activity was measured for 30 min after the saline or cocaine injections on days 1 and 7 of the study. Mice injected with cocaine had higher activity counts than those injected with saline on both days, regardless of the pretreatment (*** p

    Techniques Used: Activity Assay, Mouse Assay, Injection

    Effects of the Trk receptor antagonist on DARPP-32 levels in the lateral caudal caudate–putamen. Postweanling mice were pretreated with K252a or vehicle intracerebroventricularly 30 min before 20 mg/kg cocaine or saline i.p. for 7 d. DARPP-32 immunoreactivity was measured in the lateral caudal caudate–putamen in tissue samples obtained 18 h after the last injection. Postweanling mice that received cocaine alone (Veh/Coc) had significantly higher levels of DARPP-32 in the lateral caudal caudate–putamen than control mice (Veh/Sal). Pretreatment with the Trk receptor antagonist K252a prevented cocaine-induced upregulation of DARPP-32 (Veh/Coc vs K252a/Coc; mean ± SEM; n = 8–10/group; * p
    Figure Legend Snippet: Effects of the Trk receptor antagonist on DARPP-32 levels in the lateral caudal caudate–putamen. Postweanling mice were pretreated with K252a or vehicle intracerebroventricularly 30 min before 20 mg/kg cocaine or saline i.p. for 7 d. DARPP-32 immunoreactivity was measured in the lateral caudal caudate–putamen in tissue samples obtained 18 h after the last injection. Postweanling mice that received cocaine alone (Veh/Coc) had significantly higher levels of DARPP-32 in the lateral caudal caudate–putamen than control mice (Veh/Sal). Pretreatment with the Trk receptor antagonist K252a prevented cocaine-induced upregulation of DARPP-32 (Veh/Coc vs K252a/Coc; mean ± SEM; n = 8–10/group; * p

    Techniques Used: Mouse Assay, Injection

    39) Product Images from "Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation"

    Article Title: Fates of Neurotrophins after Retrograde Axonal Transport: Phosphorylation of p75NTR Is a Sorting Signal for Delayed Degradation

    Journal:

    doi: 10.1523/JNEUROSCI.2512-09.2009

    A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,
    Figure Legend Snippet: A–C . Conventional protein kinase C isoforms (PKCα,β,γ) effectively phosphorylate p75NTR and this phosphorylation is abolished by the kinase inhibitor K252a. A . Human p75NTR is phosphorylated in vitro by several kinases,

    Techniques Used: In Vitro

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Alomone Labs osk1 peptide
    Effect of Kv1.3 blockers on antigen-specific T cell activation. Kv1.3 blocker <t>OsK1,</t> Kv261 peptides and Kv261-HSA-34 fusion protein blocked proliferation of CD4 T cells from house dust mite allergic patients following 5 day stimulation with house dust mite extracts (A, B). IL-5 production from house dust mite-specific T cells was inhibited by Kv1.3 blockers as shown with OsK1 and Kv261-HSA-34 (C). OsK1 peptide at 0.3μM inhibited partially the proliferation of PBMC from patients allergic to ragweed after 3 day stimulation with ragweed extracts (D). Decreased proliferative response of individual donor PBMC samples by OsK1 peptide was shown in a separated graph. CTLA4-Ig or cyclosporine A (1μM) were used as reference compounds. The assays in panels A-C were repeated in 4 independent experiments with samples in duplicates. Data from one representative experiment were presented. The study in panel D was done in an experiment with 29 subjects per group. Statistical significance on the difference in proliferation between untreated and OsK1 treated groups were analyzed by 2-way ANOVA and the P value was determined and indicated as *P
    Osk1 Peptide, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/osk1 peptide/product/Alomone Labs
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    osk1 peptide - by Bioz Stars, 2022-05
    88/100 stars
      Buy from Supplier

    93
    Alomone Labs k252a
    ApoER2 proteolysis induced by NGF depends on TrkA tyrosine kinase activity and on metalloproteinase activity. PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and (A) 100 nM <t>K252a</t> or (C) with 50 μM GM6001 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2, ApoER2-CTF and the proteolytic fragment of p75 NTR (control) were determined by western blot analysis using antibodies directed against their intracellular regions. α-tubulin is shown as a loading control, and the phosphorylated form of AKT is a control for TrkA activation by NGF. (B and D) The levels of ApoER2 CTF were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, ** P
    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, 2022-05
    93/100 stars
      Buy from Supplier

    Image Search Results


    Effect of Kv1.3 blockers on antigen-specific T cell activation. Kv1.3 blocker OsK1, Kv261 peptides and Kv261-HSA-34 fusion protein blocked proliferation of CD4 T cells from house dust mite allergic patients following 5 day stimulation with house dust mite extracts (A, B). IL-5 production from house dust mite-specific T cells was inhibited by Kv1.3 blockers as shown with OsK1 and Kv261-HSA-34 (C). OsK1 peptide at 0.3μM inhibited partially the proliferation of PBMC from patients allergic to ragweed after 3 day stimulation with ragweed extracts (D). Decreased proliferative response of individual donor PBMC samples by OsK1 peptide was shown in a separated graph. CTLA4-Ig or cyclosporine A (1μM) were used as reference compounds. The assays in panels A-C were repeated in 4 independent experiments with samples in duplicates. Data from one representative experiment were presented. The study in panel D was done in an experiment with 29 subjects per group. Statistical significance on the difference in proliferation between untreated and OsK1 treated groups were analyzed by 2-way ANOVA and the P value was determined and indicated as *P

    Journal: PLoS ONE

    Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

    doi: 10.1371/journal.pone.0170102

    Figure Lengend Snippet: Effect of Kv1.3 blockers on antigen-specific T cell activation. Kv1.3 blocker OsK1, Kv261 peptides and Kv261-HSA-34 fusion protein blocked proliferation of CD4 T cells from house dust mite allergic patients following 5 day stimulation with house dust mite extracts (A, B). IL-5 production from house dust mite-specific T cells was inhibited by Kv1.3 blockers as shown with OsK1 and Kv261-HSA-34 (C). OsK1 peptide at 0.3μM inhibited partially the proliferation of PBMC from patients allergic to ragweed after 3 day stimulation with ragweed extracts (D). Decreased proliferative response of individual donor PBMC samples by OsK1 peptide was shown in a separated graph. CTLA4-Ig or cyclosporine A (1μM) were used as reference compounds. The assays in panels A-C were repeated in 4 independent experiments with samples in duplicates. Data from one representative experiment were presented. The study in panel D was done in an experiment with 29 subjects per group. Statistical significance on the difference in proliferation between untreated and OsK1 treated groups were analyzed by 2-way ANOVA and the P value was determined and indicated as *P

    Article Snippet: OsK1 peptide potently inhibited proliferation of CD4 and CD8 T cells at bead/T cell stimulation ratio of 1:1 ( ; ), but inhibition was only partial, even at maximally effective blocker concentrations.

    Techniques: Activation Assay

    Effect of Kv1.3 blockers effect on cytokine production from thapsigargin stimulated human blood and purified CD4 T cells. Effects of OsK1 and Kv261 on IL-2 production of thapsigargin stimulated CD4 T cells (A) were shown. OsK1, Kv261 peptide and Kv261-HSA-34 were tested in thapsigargin stimulated blood (B) from healthy donors and IL-2 was measured. BTP2 and cyclosporine A were used as reference compounds. The assays were performed twice in CD4 T cells and 3 times in human blood with samples in duplicates. Data from representative experiments were presented.

    Journal: PLoS ONE

    Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

    doi: 10.1371/journal.pone.0170102

    Figure Lengend Snippet: Effect of Kv1.3 blockers effect on cytokine production from thapsigargin stimulated human blood and purified CD4 T cells. Effects of OsK1 and Kv261 on IL-2 production of thapsigargin stimulated CD4 T cells (A) were shown. OsK1, Kv261 peptide and Kv261-HSA-34 were tested in thapsigargin stimulated blood (B) from healthy donors and IL-2 was measured. BTP2 and cyclosporine A were used as reference compounds. The assays were performed twice in CD4 T cells and 3 times in human blood with samples in duplicates. Data from representative experiments were presented.

    Article Snippet: OsK1 peptide potently inhibited proliferation of CD4 and CD8 T cells at bead/T cell stimulation ratio of 1:1 ( ; ), but inhibition was only partial, even at maximally effective blocker concentrations.

    Techniques: Purification

    Efficacy of Kv1.3 blocker OsK1peptide in inhibiting T cell activation is partial and dependent on stimulation strength. Effect of OsK1 peptide (filled circles) or cyclosporine A (open circles) on proliferation or IL-2 production of human primary CD4 (A—F) and CD8 T cells (G—L) activated with anti-CD3/CD28 coated beads under strong stimulation (2:1 bead:cell ratio; left panels), intermediate stimulation (1:1 bead:cell ratio; middle panels), or weak stimulation (1:2 bead:cell ratio; right panels) conditions. Cyclosporine A was not tested in CD8 T cells with weak stimulation due to insufficient purified T cells. The assays were performed in 4 independent experiments with samples in duplicates. Data from one representative experiment were presented.

    Journal: PLoS ONE

    Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

    doi: 10.1371/journal.pone.0170102

    Figure Lengend Snippet: Efficacy of Kv1.3 blocker OsK1peptide in inhibiting T cell activation is partial and dependent on stimulation strength. Effect of OsK1 peptide (filled circles) or cyclosporine A (open circles) on proliferation or IL-2 production of human primary CD4 (A—F) and CD8 T cells (G—L) activated with anti-CD3/CD28 coated beads under strong stimulation (2:1 bead:cell ratio; left panels), intermediate stimulation (1:1 bead:cell ratio; middle panels), or weak stimulation (1:2 bead:cell ratio; right panels) conditions. Cyclosporine A was not tested in CD8 T cells with weak stimulation due to insufficient purified T cells. The assays were performed in 4 independent experiments with samples in duplicates. Data from one representative experiment were presented.

    Article Snippet: OsK1 peptide potently inhibited proliferation of CD4 and CD8 T cells at bead/T cell stimulation ratio of 1:1 ( ; ), but inhibition was only partial, even at maximally effective blocker concentrations.

    Techniques: Activation Assay, Purification

    OsK1 peptide inhibited voltage-gated Kv1.3 currents in human CD4 T cells and CHO cells transfected with recombinant human Kv1.3. OsK1 peptide inhibited human Kv1.3 mediated whole cell currents in a concentration dependent manner in human CD4 T cells (A). Potency of OsK1 peptide on Kv1.3 currents in purified human CD4 T cells and Kv1.3 transfected CHO cells were determined and IC 50 curves were shown (B). Whole cell voltage-gated currents were measured in manual patch clamp assays. All recordings were made at room temperature using Multiclamp 700A amplifier and pClamp 9 software as described in Methods and Materials.

    Journal: PLoS ONE

    Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

    doi: 10.1371/journal.pone.0170102

    Figure Lengend Snippet: OsK1 peptide inhibited voltage-gated Kv1.3 currents in human CD4 T cells and CHO cells transfected with recombinant human Kv1.3. OsK1 peptide inhibited human Kv1.3 mediated whole cell currents in a concentration dependent manner in human CD4 T cells (A). Potency of OsK1 peptide on Kv1.3 currents in purified human CD4 T cells and Kv1.3 transfected CHO cells were determined and IC 50 curves were shown (B). Whole cell voltage-gated currents were measured in manual patch clamp assays. All recordings were made at room temperature using Multiclamp 700A amplifier and pClamp 9 software as described in Methods and Materials.

    Article Snippet: OsK1 peptide potently inhibited proliferation of CD4 and CD8 T cells at bead/T cell stimulation ratio of 1:1 ( ; ), but inhibition was only partial, even at maximally effective blocker concentrations.

    Techniques: Transfection, Recombinant, Concentration Assay, Purification, Patch Clamp, Software

    Effect of OsK1 on human CD4 T cell subsets. Human CD45RO - CCR7 + naïve T cells, CD45RO + CCR7 + central memory (Tcm) and CD45RO + CCR7 - effector memory (Tem) T cells were purified from human blood (A). OsK1 peptide blocked proliferation of the purified human CD4 T cell subsets induced by 2 day stimulation with anti-CD3 in the presence of PBMC (B). BTP2 and cyclosporine A were used as reference compounds in T cell assays. The assays were performed in 8 similar experiments with samples in duplicates. Data from one representative experiment were presented.

    Journal: PLoS ONE

    Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

    doi: 10.1371/journal.pone.0170102

    Figure Lengend Snippet: Effect of OsK1 on human CD4 T cell subsets. Human CD45RO - CCR7 + naïve T cells, CD45RO + CCR7 + central memory (Tcm) and CD45RO + CCR7 - effector memory (Tem) T cells were purified from human blood (A). OsK1 peptide blocked proliferation of the purified human CD4 T cell subsets induced by 2 day stimulation with anti-CD3 in the presence of PBMC (B). BTP2 and cyclosporine A were used as reference compounds in T cell assays. The assays were performed in 8 similar experiments with samples in duplicates. Data from one representative experiment were presented.

    Article Snippet: OsK1 peptide potently inhibited proliferation of CD4 and CD8 T cells at bead/T cell stimulation ratio of 1:1 ( ; ), but inhibition was only partial, even at maximally effective blocker concentrations.

    Techniques: Transmission Electron Microscopy, Purification

    ApoER2 proteolysis induced by NGF depends on TrkA tyrosine kinase activity and on metalloproteinase activity. PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and (A) 100 nM K252a or (C) with 50 μM GM6001 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2, ApoER2-CTF and the proteolytic fragment of p75 NTR (control) were determined by western blot analysis using antibodies directed against their intracellular regions. α-tubulin is shown as a loading control, and the phosphorylated form of AKT is a control for TrkA activation by NGF. (B and D) The levels of ApoER2 CTF were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, ** P

    Journal: BMC Neuroscience

    Article Title: Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway

    doi: 10.1186/1471-2202-15-108

    Figure Lengend Snippet: ApoER2 proteolysis induced by NGF depends on TrkA tyrosine kinase activity and on metalloproteinase activity. PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and (A) 100 nM K252a or (C) with 50 μM GM6001 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2, ApoER2-CTF and the proteolytic fragment of p75 NTR (control) were determined by western blot analysis using antibodies directed against their intracellular regions. α-tubulin is shown as a loading control, and the phosphorylated form of AKT is a control for TrkA activation by NGF. (B and D) The levels of ApoER2 CTF were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, ** P

    Article Snippet: For inhibitory agents, we used 100 nM K252a (Alomone Labs), 10 μM DAPT, 25 μM PD98058, 50 μM GM6001 100 nM wortmannin and 50 μM of LY294002 (all from Calbiochem) and 5 μM of ZSTK474 (Selleck Chemicals LLC) Later, the cells were treated with 100 ng/mL NGF (Alomone Lab) for different times in a 5% CO2 incubator at 37°C.

    Techniques: Activity Assay, Incubation, Western Blot, Activation Assay

    NGF induces the proteolytic processing of ApoER2. (A) Serum-starved PC12-ApoER2 cells were pre-treated with 10 μM DAPT (γ-secretase complex inhibitor), 50 μM GM6001 (metalloproteases inhibitor) and/or 100 nM K252a (Trk tyrosine kinase activity inhibitor) for 1 h and then incubated with 100 ng/mL NGF for 2 h. The blot shows full-length p75 NTR , p75 NTR CTF, and α-tubulin as a loading control. As described [ 40 ], NGF induced the proteolysis of p75 NTR and, thus, the accumulation of the CTF. This process depends on TrkA tyrosine kinase activity and the metalloproteinases. (B) Cells were pre-treated with 10 μM DAPT for 1 h and then incubated with 100 ng/mL NGF for 2 h. ApoER2 and the proteolytic fragment ApoER2-CTF were recognized using antibodies against the intracellular region of the receptor. α-tubulin is shown as a loading control. (C) Quantification of blot levels of ApoER2-CTF normalized to the loading control α-tubulin and plotted as the average ± SD of four independent experiments. Student’s t-test, ** P

    Journal: BMC Neuroscience

    Article Title: Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway

    doi: 10.1186/1471-2202-15-108

    Figure Lengend Snippet: NGF induces the proteolytic processing of ApoER2. (A) Serum-starved PC12-ApoER2 cells were pre-treated with 10 μM DAPT (γ-secretase complex inhibitor), 50 μM GM6001 (metalloproteases inhibitor) and/or 100 nM K252a (Trk tyrosine kinase activity inhibitor) for 1 h and then incubated with 100 ng/mL NGF for 2 h. The blot shows full-length p75 NTR , p75 NTR CTF, and α-tubulin as a loading control. As described [ 40 ], NGF induced the proteolysis of p75 NTR and, thus, the accumulation of the CTF. This process depends on TrkA tyrosine kinase activity and the metalloproteinases. (B) Cells were pre-treated with 10 μM DAPT for 1 h and then incubated with 100 ng/mL NGF for 2 h. ApoER2 and the proteolytic fragment ApoER2-CTF were recognized using antibodies against the intracellular region of the receptor. α-tubulin is shown as a loading control. (C) Quantification of blot levels of ApoER2-CTF normalized to the loading control α-tubulin and plotted as the average ± SD of four independent experiments. Student’s t-test, ** P

    Article Snippet: For inhibitory agents, we used 100 nM K252a (Alomone Labs), 10 μM DAPT, 25 μM PD98058, 50 μM GM6001 100 nM wortmannin and 50 μM of LY294002 (all from Calbiochem) and 5 μM of ZSTK474 (Selleck Chemicals LLC) Later, the cells were treated with 100 ng/mL NGF (Alomone Lab) for different times in a 5% CO2 incubator at 37°C.

    Techniques: Activity Assay, Incubation

    Brain-derived neurotrophic factor (BDNF) is necessary for the behavioural and neurogenic effects of oleanolic acid (OA). (A) K252a suppresses the increase of sucrose preference induced by OA administration. Control versus chronic unpredictable mild stress

    Journal: Journal of Psychiatry & Neuroscience : JPN

    Article Title: BDNF–ERK–CREB signalling mediates the role of miR-132 in the regulation of the effects of oleanolic acid in male mice

    doi: 10.1503/jpn.130169

    Figure Lengend Snippet: Brain-derived neurotrophic factor (BDNF) is necessary for the behavioural and neurogenic effects of oleanolic acid (OA). (A) K252a suppresses the increase of sucrose preference induced by OA administration. Control versus chronic unpredictable mild stress

    Article Snippet: We obtained oleanolic acid (purity = 98.4% by high- performance liquid chromatography) from Shanxi Huike Botanical Development Co., Ltd. Fluoxetine hydrochloride and bromodeoxyuridine (BrdU) were purchased from Sigma, and K252a was purchased from Alomone Laboratories.

    Techniques: Derivative Assay