k252a  (Alomone Labs)


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    Name:
    K252a
    Description:
    K252a inhibits a variety of kinases including PKC IC50 25 nM PKA IC50 18 nM and PKG IC50 20 nM It also inhibits CaMK IC50 1 8 nM and MLCK IC50 17 nM K252a has also been shown to inhibit NGF mediated proliferation through Trk receptor family
    Catalog Number:
    K-150
    Price:
    63.0
    Category:
    Small Molecule
    Source:
    Nocardiopsis sp. (soil fungi).
    Applications:
    0
    Purity:
    >98%
    Size:
    50 mcg
    Format:
    Lyophilized/solid.
    Formula:
    C27H21N3O5
    Molecular Weight:
    467.5
    Molecule Name:
    9,12-Epoxy-1H-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4-i)(1,6)benzodiazocine-10-carboxylic acid, 2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-, methyl ester, (9alpha,10beta,12alpha)-.
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    Structured Review

    Alomone Labs k252a
    K252a
    K252a inhibits a variety of kinases including PKC IC50 25 nM PKA IC50 18 nM and PKG IC50 20 nM It also inhibits CaMK IC50 1 8 nM and MLCK IC50 17 nM K252a has also been shown to inhibit NGF mediated proliferation through Trk receptor family
    https://www.bioz.com/result/k252a/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    k252a - by Bioz Stars, 2021-09
    92/100 stars

    Images

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

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

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-22-08915.2001

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

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

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

    Techniques Used: Purification, Activity Assay

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

    Techniques Used: Binding Assay, Activity Assay, Synthesized

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

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

    Related Articles

    Staining:

    Article Title: Catalpol Enhances Neurogenesis And Inhibits Apoptosis Of New Neurons Via BDNF, But Not The BDNF/Trkb Pathway
    Article Snippet: .. K252a (2.5 µg/mL, 25 µg/kg, i.p.) was obtained from Alomone Laboratories (Jerusalem, Israel), and Nissl stain was purchased from Beyotime Biotech (Jiangsu, China). ..

    other:

    Article Title: Antidepressant‐like effects of fenofibrate in mice via the hippocampal brain‐derived neurotrophic factor signalling pathway) Antidepressant‐like effects of fenofibrate in mice via the hippocampal brain‐derived neurotrophic factor signalling pathway
    Article Snippet: K252a was purchased from Alomone Laboratories (Jerusalem, Israel).

    Inhibition:

    Article Title: The receptor tyrosine kinase TrkB signals without dimerization at the plasma membrane.
    Article Snippet: .. For inhibition of dynamin and Trk activity, cells were preincubated with 100 mM Dynasore (D7693, Sigma-Aldrich) or 1 mM K252a (K-150, Alomone Labs) in poor DMEM/Neurobasal for 1 hour to allow complete blockage before stimulation with BDNF. ..

    Activity Assay:

    Article Title: The receptor tyrosine kinase TrkB signals without dimerization at the plasma membrane.
    Article Snippet: .. For inhibition of dynamin and Trk activity, cells were preincubated with 100 mM Dynasore (D7693, Sigma-Aldrich) or 1 mM K252a (K-150, Alomone Labs) in poor DMEM/Neurobasal for 1 hour to allow complete blockage before stimulation with BDNF. ..

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  • 92
    Alomone Labs anti kv1 5
    Characterization of the Kv1.3 and <t>Kv1.5</t> mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).
    Anti Kv1 5, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kv1 5/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
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    93
    Alomone Labs akap150
    Association of BMAL1 with several lipolytic machineries in primary adipocytes isolated from epididymal adipose tissues. ( A ) Direct interaction between BMAL1 and BMAL1 revealed by the co-immunoprecipitation assay. Both lysates and immunoprecipitates (IP) were subjected to immunoblotting with ( B ) anti-HSL, ( C ) anti-ATGL, ( D ) anti-Perilipin1, ( E ) <t>anti-AKAP150,</t> ( F ) anti-PKA-RIα, ( G ) anti-PKA-RIIα, and ( H ) anti-PKA-RIIβ antibodies. Relative protein expression levels of each band in E-EX and L-EX groups (levels in respective sedentary controls are set to 1). In all experiments, data are presented as the means ± S.E. ( n = 6). * p
    Akap150, 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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    93
    Alomone Labs kv1 2 antibodies
    Callosal Kv1.3 channel protein in axons and glia is altered after injury and with CFZ treatment A. Confocal overlay showing Kv1.3 ( red ) and <t>Kv1.2</t> ( green ) in rat corpus callosum 24h following midline fluid percussion TBI. Low magnification shows that each channel protein is found in reactive glia around callosal vessels (arrowheads) and along axon bundles (arrows). Inset shows paired paranodal distribution of Kv1.3 and Kv1.2 channels, some nodes with co-localization (yellow arrow), others with single channel expression (green, red arrows). B . Confocal overlays showing Kv1.3 in callosal astrocytes of sham injured (GFAP+, left-arrows; inset shows cell body and perivascular co-localization) and microglia of 24h postinjury cases (IBA1+, right-arrows). C. Western blot (WB) of protein extracts from 24h postinjury corpus callosum revealed that TBI reduced 67kD Kv1.3 levels and that CFZ treatment reversed loss of Kv1.3 expression. Data expressed as percent of paired untreated sham controls run on same blot. Lanes representative of group effects are shown in each panel. (ANOVA, *p
    Kv1 2 Antibodies, 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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    93
    Alomone Labs qx314
    Effects of <t>QX314</t> on Dopamine modulation of Glutamatergic EPSCs. A : Comparison of the average change in amplitude of the early component of the glutamatergic EPSCs (in pA) measured at −60 mV, on superfusion of DA in the presence and absence of QX314. The solid bar represents the average response in 80 neurons (n = 80), in the presence of QX314 and the dashed bar represents the average of 8 neurons (n = 8) in the absence of QX314. No statistically significant difference was found between the two groups (Paired Student’s t test p
    Qx314, 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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    Characterization of the Kv1.3 and Kv1.5 mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation *

    doi: 10.1074/jbc.M115.678995

    Figure Lengend Snippet: Characterization of the Kv1.3 and Kv1.5 mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).

    Article Snippet: Non-permeabilized cells were incubated with anti-Kv1.3 or anti Kv1.5 extracellular primary antibodies (APC101 or APC150, Alomone Labs), whereas permeabilized cells were incubated with anti-Kv1.3 COOH (75-009, NeuroMab) or anti-Kv1.5 COOH (APC004, Alomone Labs), all at a final concentration of 1:50.

    Techniques: Mutagenesis, Activation Assay, Transfection, Staining, Expressing

    Association of BMAL1 with several lipolytic machineries in primary adipocytes isolated from epididymal adipose tissues. ( A ) Direct interaction between BMAL1 and BMAL1 revealed by the co-immunoprecipitation assay. Both lysates and immunoprecipitates (IP) were subjected to immunoblotting with ( B ) anti-HSL, ( C ) anti-ATGL, ( D ) anti-Perilipin1, ( E ) anti-AKAP150, ( F ) anti-PKA-RIα, ( G ) anti-PKA-RIIα, and ( H ) anti-PKA-RIIβ antibodies. Relative protein expression levels of each band in E-EX and L-EX groups (levels in respective sedentary controls are set to 1). In all experiments, data are presented as the means ± S.E. ( n = 6). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Exercise Training-Enhanced Lipolytic Potency to Catecholamine Depends on the Time of the Day

    doi: 10.3390/ijms21186920

    Figure Lengend Snippet: Association of BMAL1 with several lipolytic machineries in primary adipocytes isolated from epididymal adipose tissues. ( A ) Direct interaction between BMAL1 and BMAL1 revealed by the co-immunoprecipitation assay. Both lysates and immunoprecipitates (IP) were subjected to immunoblotting with ( B ) anti-HSL, ( C ) anti-ATGL, ( D ) anti-Perilipin1, ( E ) anti-AKAP150, ( F ) anti-PKA-RIα, ( G ) anti-PKA-RIIα, and ( H ) anti-PKA-RIIβ antibodies. Relative protein expression levels of each band in E-EX and L-EX groups (levels in respective sedentary controls are set to 1). In all experiments, data are presented as the means ± S.E. ( n = 6). * p

    Article Snippet: This stronger potency may be due to the greater expression of lipolytic machineries expressed as fold change over the respective sedentary rats compared to their counterparts, and was accompanied by high association of BMAL1 protein with several lipolytic machineries, such as AKAP150, the regulatory subunits of PKA, HSL, and Perilipin1 proteins.

    Techniques: Isolation, Co-Immunoprecipitation Assay, Expressing

    Effect of Bmal1 expression knockdown on adipocyte lipolysis in differentiated 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were transfected with control siRNA or specific siRNA for Bmal1 using a transfection reagent. After 48 h, basal or isoproterenol-stimulated glycerol release was measured, and the cells were harvested for subsequent analysis by quantitative real-time PCR or Western blotting. ( A ) Relative expression levels of Bmal1 mRNA. The mRNA levels were measured by quantitative real-time PCR using specific primers and normalized to the amount of 18S rRNA. ( B ) Representative immunoblotting data (upper panel) and relative densities of BMAL1 protein (bottom panel) measured by Western blotting, normalized to β-actin. ( C ) Expression levels of Hsl and ATGL mRNA were measured by quantitative real-time PCR using specific primers and normalized to the amount of 18S rRNA. ( D ) Representative immunoblotting data (upper panel) and relative densities of the HSL, ATGL, and Perilipin1 proteins (lower panel) measured by Western blotting and normalized to β-actin. ( E ) Representative immunoblotting data (upper panel) and relative densities of AKAP150 protein (bottom panel) measured by Western blotting, and normalized to β-actin. ( F ) Representative immunoblotting data of PKA-RIα, -RIIα, -RIIβ, and β-actin proteins. ( G ) Relative densities of the PKA- RIα, -RIIα, and -RIIβ proteins measured by Western blotting, and normalized to β-actin. The values in the bar graphs in panels A, B, C, D, E, and G are shown relative to the mean levels detected after treatment with control siRNA, which was arbitrarily set to 1. ( H ) Reduced response of lipolysis to isoproterenol in Bmal1 -siRNA adipocytes. ( I ) Representative immunoblotting data of HSL phosphorylated at Ser563 and Ser660. ( J , K ) Relative expression levels of HSL phosphorylated at Ser563 and Ser660 as measured by Western blotting and normalized to total HSL levels. The values in the bar graphs in panels H, J, and K are shown relative to the mean level detected in basal conditions, which was arbitrarily set to 1. In all experiments, values represent the means ± S.E. of three independent experiments. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Exercise Training-Enhanced Lipolytic Potency to Catecholamine Depends on the Time of the Day

    doi: 10.3390/ijms21186920

    Figure Lengend Snippet: Effect of Bmal1 expression knockdown on adipocyte lipolysis in differentiated 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were transfected with control siRNA or specific siRNA for Bmal1 using a transfection reagent. After 48 h, basal or isoproterenol-stimulated glycerol release was measured, and the cells were harvested for subsequent analysis by quantitative real-time PCR or Western blotting. ( A ) Relative expression levels of Bmal1 mRNA. The mRNA levels were measured by quantitative real-time PCR using specific primers and normalized to the amount of 18S rRNA. ( B ) Representative immunoblotting data (upper panel) and relative densities of BMAL1 protein (bottom panel) measured by Western blotting, normalized to β-actin. ( C ) Expression levels of Hsl and ATGL mRNA were measured by quantitative real-time PCR using specific primers and normalized to the amount of 18S rRNA. ( D ) Representative immunoblotting data (upper panel) and relative densities of the HSL, ATGL, and Perilipin1 proteins (lower panel) measured by Western blotting and normalized to β-actin. ( E ) Representative immunoblotting data (upper panel) and relative densities of AKAP150 protein (bottom panel) measured by Western blotting, and normalized to β-actin. ( F ) Representative immunoblotting data of PKA-RIα, -RIIα, -RIIβ, and β-actin proteins. ( G ) Relative densities of the PKA- RIα, -RIIα, and -RIIβ proteins measured by Western blotting, and normalized to β-actin. The values in the bar graphs in panels A, B, C, D, E, and G are shown relative to the mean levels detected after treatment with control siRNA, which was arbitrarily set to 1. ( H ) Reduced response of lipolysis to isoproterenol in Bmal1 -siRNA adipocytes. ( I ) Representative immunoblotting data of HSL phosphorylated at Ser563 and Ser660. ( J , K ) Relative expression levels of HSL phosphorylated at Ser563 and Ser660 as measured by Western blotting and normalized to total HSL levels. The values in the bar graphs in panels H, J, and K are shown relative to the mean level detected in basal conditions, which was arbitrarily set to 1. In all experiments, values represent the means ± S.E. of three independent experiments. * p

    Article Snippet: This stronger potency may be due to the greater expression of lipolytic machineries expressed as fold change over the respective sedentary rats compared to their counterparts, and was accompanied by high association of BMAL1 protein with several lipolytic machineries, such as AKAP150, the regulatory subunits of PKA, HSL, and Perilipin1 proteins.

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot

    Effect of exercise training timing on the levels of lipolysis-associated proteins in primary adipocytes isolated from epididymal adipose tissues. ( A ) Representative immunoblotting data and the relative amounts of ( B ) HSL, ( C ) ATGL, ( D ) perilipin1, ( E ) CGI-58, and ( F ) PPARγ2 protein, in primary isolated adipocytes from rats, measured by Western blotting and normalized to the amount of β-actin. ( G ) Relative protein expression levels of each band in the E-EX and L-EX groups (levels in respective sedentary controls were set to 1). ( H ) Representative immunoblotting data and the relative levels of ( I ) AKAP150, ( J ) PKA-RIα, ( K ) PKA-RIIα, and ( L ) PKA-RIIβ protein measured by Western blotting and normalized to the level of β-actin. ( M ) Bmal1 mRNA expression level measured by quantitative real-time PCR and normalized to the level of 18S rRNA. In all experiments, data are presented as the means ± S.E. ( n = 6). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Exercise Training-Enhanced Lipolytic Potency to Catecholamine Depends on the Time of the Day

    doi: 10.3390/ijms21186920

    Figure Lengend Snippet: Effect of exercise training timing on the levels of lipolysis-associated proteins in primary adipocytes isolated from epididymal adipose tissues. ( A ) Representative immunoblotting data and the relative amounts of ( B ) HSL, ( C ) ATGL, ( D ) perilipin1, ( E ) CGI-58, and ( F ) PPARγ2 protein, in primary isolated adipocytes from rats, measured by Western blotting and normalized to the amount of β-actin. ( G ) Relative protein expression levels of each band in the E-EX and L-EX groups (levels in respective sedentary controls were set to 1). ( H ) Representative immunoblotting data and the relative levels of ( I ) AKAP150, ( J ) PKA-RIα, ( K ) PKA-RIIα, and ( L ) PKA-RIIβ protein measured by Western blotting and normalized to the level of β-actin. ( M ) Bmal1 mRNA expression level measured by quantitative real-time PCR and normalized to the level of 18S rRNA. In all experiments, data are presented as the means ± S.E. ( n = 6). * p

    Article Snippet: This stronger potency may be due to the greater expression of lipolytic machineries expressed as fold change over the respective sedentary rats compared to their counterparts, and was accompanied by high association of BMAL1 protein with several lipolytic machineries, such as AKAP150, the regulatory subunits of PKA, HSL, and Perilipin1 proteins.

    Techniques: Isolation, Western Blot, Expressing, Real-time Polymerase Chain Reaction

    Callosal Kv1.3 channel protein in axons and glia is altered after injury and with CFZ treatment A. Confocal overlay showing Kv1.3 ( red ) and Kv1.2 ( green ) in rat corpus callosum 24h following midline fluid percussion TBI. Low magnification shows that each channel protein is found in reactive glia around callosal vessels (arrowheads) and along axon bundles (arrows). Inset shows paired paranodal distribution of Kv1.3 and Kv1.2 channels, some nodes with co-localization (yellow arrow), others with single channel expression (green, red arrows). B . Confocal overlays showing Kv1.3 in callosal astrocytes of sham injured (GFAP+, left-arrows; inset shows cell body and perivascular co-localization) and microglia of 24h postinjury cases (IBA1+, right-arrows). C. Western blot (WB) of protein extracts from 24h postinjury corpus callosum revealed that TBI reduced 67kD Kv1.3 levels and that CFZ treatment reversed loss of Kv1.3 expression. Data expressed as percent of paired untreated sham controls run on same blot. Lanes representative of group effects are shown in each panel. (ANOVA, *p

    Journal: Experimental neurology

    Article Title: Targeting Kv1.3 channels to reduce white matter pathology after traumatic brain injury

    doi: 10.1016/j.expneurol.2016.06.011

    Figure Lengend Snippet: Callosal Kv1.3 channel protein in axons and glia is altered after injury and with CFZ treatment A. Confocal overlay showing Kv1.3 ( red ) and Kv1.2 ( green ) in rat corpus callosum 24h following midline fluid percussion TBI. Low magnification shows that each channel protein is found in reactive glia around callosal vessels (arrowheads) and along axon bundles (arrows). Inset shows paired paranodal distribution of Kv1.3 and Kv1.2 channels, some nodes with co-localization (yellow arrow), others with single channel expression (green, red arrows). B . Confocal overlays showing Kv1.3 in callosal astrocytes of sham injured (GFAP+, left-arrows; inset shows cell body and perivascular co-localization) and microglia of 24h postinjury cases (IBA1+, right-arrows). C. Western blot (WB) of protein extracts from 24h postinjury corpus callosum revealed that TBI reduced 67kD Kv1.3 levels and that CFZ treatment reversed loss of Kv1.3 expression. Data expressed as percent of paired untreated sham controls run on same blot. Lanes representative of group effects are shown in each panel. (ANOVA, *p

    Article Snippet: Post blot membranes were subjected to immunobinding with either Kv1.3 or Kv1.2 antibodies ( Alomone ; 1:100, 1:150) or IL1-β ( R & D Systems ;1:200) after published methods ( ).

    Techniques: Expressing, Western Blot

    Effects of QX314 on Dopamine modulation of Glutamatergic EPSCs. A : Comparison of the average change in amplitude of the early component of the glutamatergic EPSCs (in pA) measured at −60 mV, on superfusion of DA in the presence and absence of QX314. The solid bar represents the average response in 80 neurons (n = 80), in the presence of QX314 and the dashed bar represents the average of 8 neurons (n = 8) in the absence of QX314. No statistically significant difference was found between the two groups (Paired Student’s t test p

    Journal: PLoS ONE

    Article Title: Dopamine Preferentially Inhibits NMDA Receptor-Mediated EPSCs by Acting on Presynaptic D1 Receptors in Nucleus Accumbens during Postnatal Development

    doi: 10.1371/journal.pone.0086970

    Figure Lengend Snippet: Effects of QX314 on Dopamine modulation of Glutamatergic EPSCs. A : Comparison of the average change in amplitude of the early component of the glutamatergic EPSCs (in pA) measured at −60 mV, on superfusion of DA in the presence and absence of QX314. The solid bar represents the average response in 80 neurons (n = 80), in the presence of QX314 and the dashed bar represents the average of 8 neurons (n = 8) in the absence of QX314. No statistically significant difference was found between the two groups (Paired Student’s t test p

    Article Snippet: QX314 (5 mM; Alomone Laboratories, Jerusalem, Israel) was routinely added to the recording pipette solution to prevent voltage-sensitive Na+ channels from generating action potentials.

    Techniques: