k252a  (Alomone Labs)

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

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

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

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

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

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

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

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

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

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