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