k252a (Alomone Labs)

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Alomone Labs k252a

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

K252a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 3 article reviews
Price from $9.99 to $1999.99

k252a - by Bioz Stars, 2022-08

93/100 stars

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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

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

2) Product Images from "Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer"

Article Title: Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13181

Effects of NT inhibitor (K252a), autophagy inhibitor (CQ) and dual treatment on tumoural growth. Nude mice were subcutaneously engrafted with SW480 or SW620 (1.10 6 cells). When tumours reached a volume of 300 mm 3 , animals were divided into different groups and treated with K252a for 21 days or with CQ for 12 days or with both molecules (see Materials and methods ). ( A ) Tumour growth was determined weekly. Results are expressed in mean tumour volumes (mm 3 ) ± S.D. in comparison with the non treated group (* P

Figure Legend Snippet: Effects of NT inhibitor (K252a), autophagy inhibitor (CQ) and dual treatment on tumoural growth. Nude mice were subcutaneously engrafted with SW480 or SW620 (1.10 6 cells). When tumours reached a volume of 300 mm 3 , animals were divided into different groups and treated with K252a for 21 days or with CQ for 12 days or with both molecules (see Materials and methods ). ( A ) Tumour growth was determined weekly. Results are expressed in mean tumour volumes (mm 3 ) ± S.D. in comparison with the non treated group (* P

Techniques Used: Mouse Assay

Effects of dual treatment (K252a + CQ) on cell proliferation, cell death and vasculogenesis in tumour tissue originating from mice SW480 and SW620 xenografts. After being engrafted subcutaneously by SW480 or SW620, mice were treated by K252a + CQ as described in Materials and methods section. Tumour sample were collected from five xenograft mice for both cell lines. ( A ) Proliferation was determined by Ki67 immunostaining. Magnification was ×100. ( B ) Necrosis was quantified using HES staining. Aeras of necrosis are delimited by N. Magnification was ×50. Results are representative of the ratio between necrosis area and total tumour area obtained with the Nanozoomer Digital Pathology 2.ORS (Hamamatsu) version 2.5.88 software. ( C ) Tumour sample were lysed and total protein were extracted. VEGF and cleaved caspase 3 were assessed by Western blotting. The density of each band was calculated with ImageJ software. Images show representative results performed for each condition, for both xenografts cell lines.

Figure Legend Snippet: Effects of dual treatment (K252a + CQ) on cell proliferation, cell death and vasculogenesis in tumour tissue originating from mice SW480 and SW620 xenografts. After being engrafted subcutaneously by SW480 or SW620, mice were treated by K252a + CQ as described in Materials and methods section. Tumour sample were collected from five xenograft mice for both cell lines. ( A ) Proliferation was determined by Ki67 immunostaining. Magnification was ×100. ( B ) Necrosis was quantified using HES staining. Aeras of necrosis are delimited by N. Magnification was ×50. Results are representative of the ratio between necrosis area and total tumour area obtained with the Nanozoomer Digital Pathology 2.ORS (Hamamatsu) version 2.5.88 software. ( C ) Tumour sample were lysed and total protein were extracted. VEGF and cleaved caspase 3 were assessed by Western blotting. The density of each band was calculated with ImageJ software. Images show representative results performed for each condition, for both xenografts cell lines.

Techniques Used: Mouse Assay, Immunostaining, Staining, Digital Pathology, Software, Western Blot

Metabolic activity, cell death analysis and PARP cleavage in CRC cell lines after K252a, CQ and K252a + CQ treatments. SW480 and SW620 were treated with K252a, CQ or both molecules for 72 hrs (see Materials and methods ). ( A ) Metabolic activity was assessed through MTT testing. Histograms show the means ± S.E.M. of at least three independent experiments (* P

Figure Legend Snippet: Metabolic activity, cell death analysis and PARP cleavage in CRC cell lines after K252a, CQ and K252a + CQ treatments. SW480 and SW620 were treated with K252a, CQ or both molecules for 72 hrs (see Materials and methods ). ( A ) Metabolic activity was assessed through MTT testing. Histograms show the means ± S.E.M. of at least three independent experiments (* P

Techniques Used: Activity Assay, MTT Assay

Effect of tyrosine kinase receptor inhibitor (K252a) on TrkB/BDNF expression and autophagy induction. SW480 and SW620 were treated with 100 nM of K252a for 3 hrs, recovered, washed and total RNA and proteins were extracted (see Materials and methods ). ( A ) BDNF, Full Length (FL) and Truncated (Tr) TrkB, transcripts expression was assessed by qPCR. Histograms are representative of three independent experiments. The fold expression was obtained by the comparative cycle threshold method using the GAPDH RNA expression level as internal control. ( B ) BDNF, FL TrkB, Tr TrkB, phospho‐TrkB (P TrkB), AKT, phospho‐AKT (P AKT), mTOR and phospho‐mTOR (P mTOR), proteins expression were assessed by Western blotting. The density of each band representative for protein expression was calculated with ImageJ software. Images show representative results of three experiments. Statistically results are explained in comparison with control cells, normalized at 1 (* P

Figure Legend Snippet: Effect of tyrosine kinase receptor inhibitor (K252a) on TrkB/BDNF expression and autophagy induction. SW480 and SW620 were treated with 100 nM of K252a for 3 hrs, recovered, washed and total RNA and proteins were extracted (see Materials and methods ). ( A ) BDNF, Full Length (FL) and Truncated (Tr) TrkB, transcripts expression was assessed by qPCR. Histograms are representative of three independent experiments. The fold expression was obtained by the comparative cycle threshold method using the GAPDH RNA expression level as internal control. ( B ) BDNF, FL TrkB, Tr TrkB, phospho‐TrkB (P TrkB), AKT, phospho‐AKT (P AKT), mTOR and phospho‐mTOR (P mTOR), proteins expression were assessed by Western blotting. The density of each band representative for protein expression was calculated with ImageJ software. Images show representative results of three experiments. Statistically results are explained in comparison with control cells, normalized at 1 (* P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, RNA Expression, Western Blot, Software

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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.

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.

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.

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.

Techniques: Transmission Electron Microscopy, Purification

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

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

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

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: