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    ProSci Incorporated rabbit anti rat s1pr1 antibody
    Hippocampal sphingosine 1-phosphate receptor 1 <t>(S1PR1)</t> expression increased markedly after TBI. (a) The level of S1PR1 protein in hippocampus was detected by western blotting (WB) at 12 hours, 1, 3, 7, 14, 21, and 28 days after trauma. (b) Quantitative analysis indicated that upregulated expression of hippocampal S1PR1 occurred from 12 hours, peaked at 7 days, and remained at a relatively higher level for at least 28 days after TBI ( n = 3, sham group; n = 5 each time-point group of TBI). Further analysis revealed that there was a statistical difference between any two time-point groups after trauma. ∗ P < 0.05 versus sham group; # P < 0.05 versus other time-point groups of TBI.
    Rabbit Anti Rat S1pr1 Antibody, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    MedChemExpress sphingosine-1-phosphate
    Hippocampal sphingosine 1-phosphate receptor 1 <t>(S1PR1)</t> expression increased markedly after TBI. (a) The level of S1PR1 protein in hippocampus was detected by western blotting (WB) at 12 hours, 1, 3, 7, 14, 21, and 28 days after trauma. (b) Quantitative analysis indicated that upregulated expression of hippocampal S1PR1 occurred from 12 hours, peaked at 7 days, and remained at a relatively higher level for at least 28 days after TBI ( n = 3, sham group; n = 5 each time-point group of TBI). Further analysis revealed that there was a statistical difference between any two time-point groups after trauma. ∗ P < 0.05 versus sham group; # P < 0.05 versus other time-point groups of TBI.
    Sphingosine 1 Phosphate, supplied by MedChemExpress, 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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    85
    Alomone Labs rabbit polyclonal anti s1p 2 antibody
    <t>S1P</t> receptor antagonists attenuate phosphorylation responses of ERK1/2 in HUVEC induced by COA-Cl and S1P. The figure shows the results of immunoblot (IB) analyses in which HUVEC were treated with COA-Cl or S1P either in the presence or absence of pretreatment with antagonists specific for S1P receptors. (A) Effects of the S1P 1 antagonist W146. HUVEC were treated with 10 μ mol/L of W146 for 30 min, followed by 100 μ mol/L of COA-Cl for 15 min. They were then subjected to IB analyses for phospho- and total-ERK1/2. The upper half of the panel shows the representative results of four independent experiments, which yielded equivalent data. The results are summarized in the lower half graphs. (B) VPC23019, a dual antagonist for S1P 1 /S1P 3 (5 μ mol/L for 15 min), was used instead of W146 ( n = 6). (C and D) Cells were treated with 1 μ mol/L of S1P for 5 min instead of COA-Cl ( n = 4), respectively. * P < 0.05 versus vehicle alone. † P < 0.05 versus cells not treated with W146 or VPC23019.
    Rabbit Polyclonal Anti S1p 2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Avanti Polar sphingosine-1-phosphate
    <t>S1P</t> receptor antagonists attenuate phosphorylation responses of ERK1/2 in HUVEC induced by COA-Cl and S1P. The figure shows the results of immunoblot (IB) analyses in which HUVEC were treated with COA-Cl or S1P either in the presence or absence of pretreatment with antagonists specific for S1P receptors. (A) Effects of the S1P 1 antagonist W146. HUVEC were treated with 10 μ mol/L of W146 for 30 min, followed by 100 μ mol/L of COA-Cl for 15 min. They were then subjected to IB analyses for phospho- and total-ERK1/2. The upper half of the panel shows the representative results of four independent experiments, which yielded equivalent data. The results are summarized in the lower half graphs. (B) VPC23019, a dual antagonist for S1P 1 /S1P 3 (5 μ mol/L for 15 min), was used instead of W146 ( n = 6). (C and D) Cells were treated with 1 μ mol/L of S1P for 5 min instead of COA-Cl ( n = 4), respectively. * P < 0.05 versus vehicle alone. † P < 0.05 versus cells not treated with W146 or VPC23019.
    Sphingosine 1 Phosphate, supplied by Avanti Polar, 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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    86
    Millipore sphingosine 1 phosphate s 1p
    shCTL or shRGS13 cells were loaded with a calcium-sensing dye, follwed by stimulation with various concentrations of adenosine (A), C5a (B), CXCL12 (C), 100 PM sphingosine 1-phosphate (S-1P) (D) or ionomycin (5 μM, E) and measurement of intracellular Ca2+ concentration by fluorimetry. Each data point represents the mean ± S.E.M. (maximum–minimum value of relative fluorescence units [RFU]) obtained from 3-4 independent experiments (*P=0.004, 1-way ANOVA). Representative kinetic tracings of intracellular Ca2+ after stimulation with each agonist are shown on the right. Time of stimulus addition is indicated by an arrow.
    Sphingosine 1 Phosphate S 1p, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Hippocampal sphingosine 1-phosphate receptor 1 (S1PR1) expression increased markedly after TBI. (a) The level of S1PR1 protein in hippocampus was detected by western blotting (WB) at 12 hours, 1, 3, 7, 14, 21, and 28 days after trauma. (b) Quantitative analysis indicated that upregulated expression of hippocampal S1PR1 occurred from 12 hours, peaked at 7 days, and remained at a relatively higher level for at least 28 days after TBI ( n = 3, sham group; n = 5 each time-point group of TBI). Further analysis revealed that there was a statistical difference between any two time-point groups after trauma. ∗ P < 0.05 versus sham group; # P < 0.05 versus other time-point groups of TBI.

    Journal: Neural Plasticity

    Article Title: Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

    doi: 10.1155/2016/8072156

    Figure Lengend Snippet: Hippocampal sphingosine 1-phosphate receptor 1 (S1PR1) expression increased markedly after TBI. (a) The level of S1PR1 protein in hippocampus was detected by western blotting (WB) at 12 hours, 1, 3, 7, 14, 21, and 28 days after trauma. (b) Quantitative analysis indicated that upregulated expression of hippocampal S1PR1 occurred from 12 hours, peaked at 7 days, and remained at a relatively higher level for at least 28 days after TBI ( n = 3, sham group; n = 5 each time-point group of TBI). Further analysis revealed that there was a statistical difference between any two time-point groups after trauma. ∗ P < 0.05 versus sham group; # P < 0.05 versus other time-point groups of TBI.

    Article Snippet: Following blocked by 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20 (TBST), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-rat S1PR1 antibody (1 : 500, Prosci, 4809, Poway, CA, USA), rabbit anti-rat phosphate Erk (pErk) antibody (1 : 1000, Cell Signaling, 9101, Beverly, MA, USA), rabbit anti-rat total Erk (tErk) antibody (1 : 1000, Cell Signaling, 9102, Beverly, MA, USA), rabbit anti-rat phosphate MEK (pMEK) antibody (1 : 1000, Cell Signaling, 9154, Beverly, MA, USA), and rabbit anti-rat total MEK (tMEK) antibody (1 : 1000, Cell Signaling, 9126, Beverly, MA, USA).

    Techniques: Expressing, Western Blot

    S1PR1 activation enhanced NSCs proliferation and neuronal differentiation in hippocampal dentate gyrus (DG) after TBI. (a) Coronal diagram of rat hippocampus, the subgranular zone, granular cells layer, and molecular layer of DG were, respectively, marked by SGZ, GCL, and MOL, the blue pane representing one of microscopic visual fields for counting immunolabeled cells of immunofluorescence (IF). (b) NSCs proliferation in SGZ at 7 days after TBI was assessed by BrdU/SOX2 double-labeling IF. Quantitation analysis showed that, relative to sham group ( n = 3), brain injury induced more double-positive cells in TBI+Vehicle group ( n = 6). Following the treatment of SEW2871, the number of BrdU + /SOX2 + cells further increased in TBI+SEW group ( n = 6). Reversely, administration of VPC23019 resulted in a significant reduction of BrdU + /SOX2 + cells in TBI+SEW+VPC group ( n = 6). (c) Neuronal differentiation of NSCs in SGZ at 28 days after TBI was assessed by BrdU/NeuN double-labeling IF. Statistical data indicated that BrdU + /NeuN + cells increased in TBI+Vehicle group ( n = 6) compared with sham group ( n = 3). And the double-positive cells increased at even higher level after SEW2871 treatment in TBI+SEW group ( n = 6). However, VPC23019 administration caused an evident decrease of BrdU + /NeuN + cells in TBI+SEW+VPC group ( n = 6). (d) and (e) Representative IF microphotographs of hippocampus immunolabeled with BrdU/SOX2 and BrdU/NeuN in all the above four groups at 7 days and 28 days after trauma. Scale bar: 50 μ m. ∗ P < 0.05 versus sham group; # P < 0.05 versus TBI+Vehicle group or TBI+SEW+VPC group.

    Journal: Neural Plasticity

    Article Title: Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

    doi: 10.1155/2016/8072156

    Figure Lengend Snippet: S1PR1 activation enhanced NSCs proliferation and neuronal differentiation in hippocampal dentate gyrus (DG) after TBI. (a) Coronal diagram of rat hippocampus, the subgranular zone, granular cells layer, and molecular layer of DG were, respectively, marked by SGZ, GCL, and MOL, the blue pane representing one of microscopic visual fields for counting immunolabeled cells of immunofluorescence (IF). (b) NSCs proliferation in SGZ at 7 days after TBI was assessed by BrdU/SOX2 double-labeling IF. Quantitation analysis showed that, relative to sham group ( n = 3), brain injury induced more double-positive cells in TBI+Vehicle group ( n = 6). Following the treatment of SEW2871, the number of BrdU + /SOX2 + cells further increased in TBI+SEW group ( n = 6). Reversely, administration of VPC23019 resulted in a significant reduction of BrdU + /SOX2 + cells in TBI+SEW+VPC group ( n = 6). (c) Neuronal differentiation of NSCs in SGZ at 28 days after TBI was assessed by BrdU/NeuN double-labeling IF. Statistical data indicated that BrdU + /NeuN + cells increased in TBI+Vehicle group ( n = 6) compared with sham group ( n = 3). And the double-positive cells increased at even higher level after SEW2871 treatment in TBI+SEW group ( n = 6). However, VPC23019 administration caused an evident decrease of BrdU + /NeuN + cells in TBI+SEW+VPC group ( n = 6). (d) and (e) Representative IF microphotographs of hippocampus immunolabeled with BrdU/SOX2 and BrdU/NeuN in all the above four groups at 7 days and 28 days after trauma. Scale bar: 50 μ m. ∗ P < 0.05 versus sham group; # P < 0.05 versus TBI+Vehicle group or TBI+SEW+VPC group.

    Article Snippet: Following blocked by 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20 (TBST), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-rat S1PR1 antibody (1 : 500, Prosci, 4809, Poway, CA, USA), rabbit anti-rat phosphate Erk (pErk) antibody (1 : 1000, Cell Signaling, 9101, Beverly, MA, USA), rabbit anti-rat total Erk (tErk) antibody (1 : 1000, Cell Signaling, 9102, Beverly, MA, USA), rabbit anti-rat phosphate MEK (pMEK) antibody (1 : 1000, Cell Signaling, 9154, Beverly, MA, USA), and rabbit anti-rat total MEK (tMEK) antibody (1 : 1000, Cell Signaling, 9126, Beverly, MA, USA).

    Techniques: Activation Assay, Immunolabeling, Immunofluorescence, Labeling, Quantitation Assay

    Activation of S1PR1 triggered MEK/Erk pathway phosphorylation in hippocampus at 7 days after TBI. (a) WB was performed to determine hippocampal S1PR1, pMEK, tMEK, pErk, and tErk expression in groups of sham, TBI+Vehicle, TBI+SEW, and TBI+SEW+VPC group ( n = 3 in sham and n = 6 in other groups). (b, c, and e) The level of S1PR1, pMEK, and pErk significantly increased after trauma. SEW2871 induced further upregulation of S1PR1, pMEK, and pErk. However, the effect was attenuated by administration of VPC23019. (d, f) Neither S1PR1 agonism nor antagonism had effect on tMEK and tErk expression in hippocampus after TBI. ∗ P < 0.05 versus sham group; # P < 0.05 versus TBI+Vehicle group or TBI+SEW+VPC group.

    Journal: Neural Plasticity

    Article Title: Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

    doi: 10.1155/2016/8072156

    Figure Lengend Snippet: Activation of S1PR1 triggered MEK/Erk pathway phosphorylation in hippocampus at 7 days after TBI. (a) WB was performed to determine hippocampal S1PR1, pMEK, tMEK, pErk, and tErk expression in groups of sham, TBI+Vehicle, TBI+SEW, and TBI+SEW+VPC group ( n = 3 in sham and n = 6 in other groups). (b, c, and e) The level of S1PR1, pMEK, and pErk significantly increased after trauma. SEW2871 induced further upregulation of S1PR1, pMEK, and pErk. However, the effect was attenuated by administration of VPC23019. (d, f) Neither S1PR1 agonism nor antagonism had effect on tMEK and tErk expression in hippocampus after TBI. ∗ P < 0.05 versus sham group; # P < 0.05 versus TBI+Vehicle group or TBI+SEW+VPC group.

    Article Snippet: Following blocked by 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20 (TBST), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-rat S1PR1 antibody (1 : 500, Prosci, 4809, Poway, CA, USA), rabbit anti-rat phosphate Erk (pErk) antibody (1 : 1000, Cell Signaling, 9101, Beverly, MA, USA), rabbit anti-rat total Erk (tErk) antibody (1 : 1000, Cell Signaling, 9102, Beverly, MA, USA), rabbit anti-rat phosphate MEK (pMEK) antibody (1 : 1000, Cell Signaling, 9154, Beverly, MA, USA), and rabbit anti-rat total MEK (tMEK) antibody (1 : 1000, Cell Signaling, 9126, Beverly, MA, USA).

    Techniques: Activation Assay, Expressing

    S1PR1-induced NSCs proliferation and neuronal differentiation in TBI rats were affected by MEK/Erk activity. (a, b) Representative IF microphotographs of hippocampus immunolabeled with BrdU/SOX2 and BrdU/NeuN in TBI+SEW, TBI+SEW+U0126, TBI+VPC, and TBI+VPC+ERN groups at 7 days and 28 days after trauma ( n = 6 in each group). (c, d) Statistical analysis showed that, compared to TBI+SEW group, administration of U0126 significantly decreased the number of BrdU+/SOX2+ cells and BrdU+/NeuN+ cells in TBI+SEW+U0126 group. Reversely, the double-labeled cells in TBI+VPC+ERN group were significantly increased compared with TBI+VPC group. Scale bar: 50 μ m. ∗ P < 0.05 versus TBI+SEW group; # P < 0.05 versus TBI+VPC group.

    Journal: Neural Plasticity

    Article Title: Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

    doi: 10.1155/2016/8072156

    Figure Lengend Snippet: S1PR1-induced NSCs proliferation and neuronal differentiation in TBI rats were affected by MEK/Erk activity. (a, b) Representative IF microphotographs of hippocampus immunolabeled with BrdU/SOX2 and BrdU/NeuN in TBI+SEW, TBI+SEW+U0126, TBI+VPC, and TBI+VPC+ERN groups at 7 days and 28 days after trauma ( n = 6 in each group). (c, d) Statistical analysis showed that, compared to TBI+SEW group, administration of U0126 significantly decreased the number of BrdU+/SOX2+ cells and BrdU+/NeuN+ cells in TBI+SEW+U0126 group. Reversely, the double-labeled cells in TBI+VPC+ERN group were significantly increased compared with TBI+VPC group. Scale bar: 50 μ m. ∗ P < 0.05 versus TBI+SEW group; # P < 0.05 versus TBI+VPC group.

    Article Snippet: Following blocked by 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20 (TBST), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-rat S1PR1 antibody (1 : 500, Prosci, 4809, Poway, CA, USA), rabbit anti-rat phosphate Erk (pErk) antibody (1 : 1000, Cell Signaling, 9101, Beverly, MA, USA), rabbit anti-rat total Erk (tErk) antibody (1 : 1000, Cell Signaling, 9102, Beverly, MA, USA), rabbit anti-rat phosphate MEK (pMEK) antibody (1 : 1000, Cell Signaling, 9154, Beverly, MA, USA), and rabbit anti-rat total MEK (tMEK) antibody (1 : 1000, Cell Signaling, 9126, Beverly, MA, USA).

    Techniques: Activity Assay, Immunolabeling, Labeling

    Cognitive function of rats in sham, TBI+Vehicle, TBI+SEW, TBI+SEW+U0126, TBI+VPC, and TBI+VPC+ERN group ( n = 3 in sham and n = 5 in other groups) were evaluated by Morris water maze (MWM) test. (a) Escape latency in hidden platform trial exhibited a gradual reduction tendency from 24 to 27 days after trauma. Daily escape latency of TBI+Vehicle group was longer than that of sham group. Use of S1PR1 agonist in TBI+SEW group significantly shorten the latency, but the effect was eliminated in TBI+SEW+U0126 group. In addition, the escape latency of TBI+VPC+ERN group decreased compared with TBI+VPC group. (b, c) Platform crossing times and target quadrant duration in probe trial revealed that, relative to sham group, TBI+Vehicle group presented less times and shorter duration at 28 days after TBI. S1PR1 activation significantly increased the two indexes of TBI+SEW group, but the favorable effect was blocked by U0126 treatment in TBI+SEW+U0126 group. Moreover, the times of platform crossing and duration in target quadrant of TBI+VPC group were lower than those of TBI+VPC+ERN group. (d) Rat swimming speed of the six groups did not show any statistical difference. ∗ P < 0.05 versus sham group; # P < 0.05 versus TBI+SEW group, † P < 0.05 versus TBI+VPC group.

    Journal: Neural Plasticity

    Article Title: Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

    doi: 10.1155/2016/8072156

    Figure Lengend Snippet: Cognitive function of rats in sham, TBI+Vehicle, TBI+SEW, TBI+SEW+U0126, TBI+VPC, and TBI+VPC+ERN group ( n = 3 in sham and n = 5 in other groups) were evaluated by Morris water maze (MWM) test. (a) Escape latency in hidden platform trial exhibited a gradual reduction tendency from 24 to 27 days after trauma. Daily escape latency of TBI+Vehicle group was longer than that of sham group. Use of S1PR1 agonist in TBI+SEW group significantly shorten the latency, but the effect was eliminated in TBI+SEW+U0126 group. In addition, the escape latency of TBI+VPC+ERN group decreased compared with TBI+VPC group. (b, c) Platform crossing times and target quadrant duration in probe trial revealed that, relative to sham group, TBI+Vehicle group presented less times and shorter duration at 28 days after TBI. S1PR1 activation significantly increased the two indexes of TBI+SEW group, but the favorable effect was blocked by U0126 treatment in TBI+SEW+U0126 group. Moreover, the times of platform crossing and duration in target quadrant of TBI+VPC group were lower than those of TBI+VPC+ERN group. (d) Rat swimming speed of the six groups did not show any statistical difference. ∗ P < 0.05 versus sham group; # P < 0.05 versus TBI+SEW group, † P < 0.05 versus TBI+VPC group.

    Article Snippet: Following blocked by 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20 (TBST), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-rat S1PR1 antibody (1 : 500, Prosci, 4809, Poway, CA, USA), rabbit anti-rat phosphate Erk (pErk) antibody (1 : 1000, Cell Signaling, 9101, Beverly, MA, USA), rabbit anti-rat total Erk (tErk) antibody (1 : 1000, Cell Signaling, 9102, Beverly, MA, USA), rabbit anti-rat phosphate MEK (pMEK) antibody (1 : 1000, Cell Signaling, 9154, Beverly, MA, USA), and rabbit anti-rat total MEK (tMEK) antibody (1 : 1000, Cell Signaling, 9126, Beverly, MA, USA).

    Techniques: Activation Assay

    S1P receptor antagonists attenuate phosphorylation responses of ERK1/2 in HUVEC induced by COA-Cl and S1P. The figure shows the results of immunoblot (IB) analyses in which HUVEC were treated with COA-Cl or S1P either in the presence or absence of pretreatment with antagonists specific for S1P receptors. (A) Effects of the S1P 1 antagonist W146. HUVEC were treated with 10 μ mol/L of W146 for 30 min, followed by 100 μ mol/L of COA-Cl for 15 min. They were then subjected to IB analyses for phospho- and total-ERK1/2. The upper half of the panel shows the representative results of four independent experiments, which yielded equivalent data. The results are summarized in the lower half graphs. (B) VPC23019, a dual antagonist for S1P 1 /S1P 3 (5 μ mol/L for 15 min), was used instead of W146 ( n = 6). (C and D) Cells were treated with 1 μ mol/L of S1P for 5 min instead of COA-Cl ( n = 4), respectively. * P < 0.05 versus vehicle alone. † P < 0.05 versus cells not treated with W146 or VPC23019.

    Journal: Pharmacology Research & Perspectives

    Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

    doi: 10.1002/prp2.68

    Figure Lengend Snippet: S1P receptor antagonists attenuate phosphorylation responses of ERK1/2 in HUVEC induced by COA-Cl and S1P. The figure shows the results of immunoblot (IB) analyses in which HUVEC were treated with COA-Cl or S1P either in the presence or absence of pretreatment with antagonists specific for S1P receptors. (A) Effects of the S1P 1 antagonist W146. HUVEC were treated with 10 μ mol/L of W146 for 30 min, followed by 100 μ mol/L of COA-Cl for 15 min. They were then subjected to IB analyses for phospho- and total-ERK1/2. The upper half of the panel shows the representative results of four independent experiments, which yielded equivalent data. The results are summarized in the lower half graphs. (B) VPC23019, a dual antagonist for S1P 1 /S1P 3 (5 μ mol/L for 15 min), was used instead of W146 ( n = 6). (C and D) Cells were treated with 1 μ mol/L of S1P for 5 min instead of COA-Cl ( n = 4), respectively. * P < 0.05 versus vehicle alone. † P < 0.05 versus cells not treated with W146 or VPC23019.

    Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

    Techniques: Western Blot

    Immunoblot assay and RT-PCR analysis of HUVEC for S1P receptor subtypes and effects of siRNA. (A) Results of immunoblot (IB) analyses using lysates derived from HUVEC and tissue homogenates derived from healthy male rats. Equal quantities of cellular proteins were separated and probed using antibodies directed to S1P receptor subtypes S1P 1–3 or to MAP kinases ERK1/2, as indicated. HUVEC expressed abundant S1P 1 and detectable S1P 3 immunoreactive signals but did not express S1P 2 ( n = 3). (B) Effective knockdown of S1P receptor proteins in HUVEC transiently transfected with siRNA directed to S1P 1 or S1P 3 . In the left half of the figure, the cells were transfected with siRNA specific for S1P 1 (Qiagen Hs_EDG1_1, 40 nmol/L) or negative control siRNA. Forty-eight hours after transfection, the cells were harvested and subjected to a series of IB assays using antibodies directed to S1P 1 , S1P 3 , ERK1/2, VEGFR2, GAPDH and cyclophilin-B (Cy-B). In the right half, the cells were transfected with siRNA specific for S1P 3 (Qiagen Hs_EDG3_6, 20 nmol/L) or control siRNA. For both halves, six independent cultures were tested that yielded similar results. siRNA directed to S1P 1 or S1P 3 leads to significant and specific knockdown of their target protein. (C) Results of RT-PCR assays. HUVEC were transiently transfected with Qiagen siRNA directed to human S1P 1 receptor or with negative control. They were then subjected to RNA isolation and RT-PCR assays using primer pairs as indicated. Note that these cells express detectable levels of S1P receptor subtypes S1P 1−3 along with VEGFR2 and GAPDH. Densitometric analysis revealed that the expression level of S1P 1 transcript relative to GAPDH in cells treated with siRNA specific to S1P 1 decreased to 24.5% ± 5.5% of the control, while those of S1P 3 , S1P 2 , and VEGFR2 remained unchanged ( n = 3).

    Journal: Pharmacology Research & Perspectives

    Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

    doi: 10.1002/prp2.68

    Figure Lengend Snippet: Immunoblot assay and RT-PCR analysis of HUVEC for S1P receptor subtypes and effects of siRNA. (A) Results of immunoblot (IB) analyses using lysates derived from HUVEC and tissue homogenates derived from healthy male rats. Equal quantities of cellular proteins were separated and probed using antibodies directed to S1P receptor subtypes S1P 1–3 or to MAP kinases ERK1/2, as indicated. HUVEC expressed abundant S1P 1 and detectable S1P 3 immunoreactive signals but did not express S1P 2 ( n = 3). (B) Effective knockdown of S1P receptor proteins in HUVEC transiently transfected with siRNA directed to S1P 1 or S1P 3 . In the left half of the figure, the cells were transfected with siRNA specific for S1P 1 (Qiagen Hs_EDG1_1, 40 nmol/L) or negative control siRNA. Forty-eight hours after transfection, the cells were harvested and subjected to a series of IB assays using antibodies directed to S1P 1 , S1P 3 , ERK1/2, VEGFR2, GAPDH and cyclophilin-B (Cy-B). In the right half, the cells were transfected with siRNA specific for S1P 3 (Qiagen Hs_EDG3_6, 20 nmol/L) or control siRNA. For both halves, six independent cultures were tested that yielded similar results. siRNA directed to S1P 1 or S1P 3 leads to significant and specific knockdown of their target protein. (C) Results of RT-PCR assays. HUVEC were transiently transfected with Qiagen siRNA directed to human S1P 1 receptor or with negative control. They were then subjected to RNA isolation and RT-PCR assays using primer pairs as indicated. Note that these cells express detectable levels of S1P receptor subtypes S1P 1−3 along with VEGFR2 and GAPDH. Densitometric analysis revealed that the expression level of S1P 1 transcript relative to GAPDH in cells treated with siRNA specific to S1P 1 decreased to 24.5% ± 5.5% of the control, while those of S1P 3 , S1P 2 , and VEGFR2 remained unchanged ( n = 3).

    Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Transfection, Negative Control, Isolation, Expressing

    Effects of siRNA directed to S1P receptors on HUVEC stimulated with COA-Cl or S1P. The results of immunoblot (IB) analyses using lysates derived from HUVEC treated with COA-Cl or S1P that had been transfected with siRNA directed to S1P receptor subtypes are shown. (A) HUVEC transfected with siRNA specific for S1P 1 or control oligonucleotides were treated with COA-Cl (100 μ mol/L, upper half) for the times indicated, followed by IB analyses for phosphorylated and total forms of ERK1/2. (B) Transfection was performed using siRNA specific for S1P 3 . (C and D) Cells were treated with S1P (100 nmol/L) instead of COA-Cl ( n = 4) for each subset. * P < 0.05 versus vehicle alone. † P < 0.05 versus control siRNA.

    Journal: Pharmacology Research & Perspectives

    Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

    doi: 10.1002/prp2.68

    Figure Lengend Snippet: Effects of siRNA directed to S1P receptors on HUVEC stimulated with COA-Cl or S1P. The results of immunoblot (IB) analyses using lysates derived from HUVEC treated with COA-Cl or S1P that had been transfected with siRNA directed to S1P receptor subtypes are shown. (A) HUVEC transfected with siRNA specific for S1P 1 or control oligonucleotides were treated with COA-Cl (100 μ mol/L, upper half) for the times indicated, followed by IB analyses for phosphorylated and total forms of ERK1/2. (B) Transfection was performed using siRNA specific for S1P 3 . (C and D) Cells were treated with S1P (100 nmol/L) instead of COA-Cl ( n = 4) for each subset. * P < 0.05 versus vehicle alone. † P < 0.05 versus control siRNA.

    Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

    Techniques: Western Blot, Derivative Assay, Transfection

    Promotion of tube formation activity by COA-Cl in HUVEC and inhibitory effects of S1P 1 antagonists and a Src tyrosine kinase. The results of tube formation analyses are shown. (A) HUVEC were cocultured with normal human fibroblasts for 10 days in the presence or absence of COA-Cl (100 μ mol/L), with/without W146 (20 μ mol/L) or VPC23019 (5 μ mol/L), as indicated. Endothelial tube formation was then identified as CD31-positive signals in microphotographs, which were subsequently quantified in the graph. The right half of the figure summarizes the results derived from four independent assays, representative images of which are shown in the left half. (B) Certain cells were treated with PP2 (2.5 μ mol/L). In both panels, scale bars correspond to 500 micrometer ( n = 4).

    Journal: Pharmacology Research & Perspectives

    Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

    doi: 10.1002/prp2.68

    Figure Lengend Snippet: Promotion of tube formation activity by COA-Cl in HUVEC and inhibitory effects of S1P 1 antagonists and a Src tyrosine kinase. The results of tube formation analyses are shown. (A) HUVEC were cocultured with normal human fibroblasts for 10 days in the presence or absence of COA-Cl (100 μ mol/L), with/without W146 (20 μ mol/L) or VPC23019 (5 μ mol/L), as indicated. Endothelial tube formation was then identified as CD31-positive signals in microphotographs, which were subsequently quantified in the graph. The right half of the figure summarizes the results derived from four independent assays, representative images of which are shown in the left half. (B) Certain cells were treated with PP2 (2.5 μ mol/L). In both panels, scale bars correspond to 500 micrometer ( n = 4).

    Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

    Techniques: Activity Assay, Derivative Assay

    Displacement of [ 3 H]S1P by COA-Cl in receptor ligand-binding competition assay using membrane preparation derived from S1P 1 -overexpressing chem-1 cells. The figure shows the results of receptor ligand-binding competition assay in which [ 3 H]S1P was bound to membrane preparation derived from chem-1 cells overexpressing S1P 1 . Increasing concentrations of COA-Cl, unlabeled S1P, or unlabeled adenosine were included along with [ 3 H]S1P (A−C, respectively). In each panel, 100% and 0% correspond to the [ 3 H]S1P binding values obtained in the presence of the vehicle and excess S1P, respectively ( n = 6−9). (D) Degrees of [ 3 H]S1P binding obtained in the presence of the vehicle, COA-Cl (500 μ mol/L), S1P (10 μ mol/L) and adenosine (2.5 mmol/L). COA-Cl displaced [ 3 H]S1P but adenosine did not.

    Journal: Pharmacology Research & Perspectives

    Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

    doi: 10.1002/prp2.68

    Figure Lengend Snippet: Displacement of [ 3 H]S1P by COA-Cl in receptor ligand-binding competition assay using membrane preparation derived from S1P 1 -overexpressing chem-1 cells. The figure shows the results of receptor ligand-binding competition assay in which [ 3 H]S1P was bound to membrane preparation derived from chem-1 cells overexpressing S1P 1 . Increasing concentrations of COA-Cl, unlabeled S1P, or unlabeled adenosine were included along with [ 3 H]S1P (A−C, respectively). In each panel, 100% and 0% correspond to the [ 3 H]S1P binding values obtained in the presence of the vehicle and excess S1P, respectively ( n = 6−9). (D) Degrees of [ 3 H]S1P binding obtained in the presence of the vehicle, COA-Cl (500 μ mol/L), S1P (10 μ mol/L) and adenosine (2.5 mmol/L). COA-Cl displaced [ 3 H]S1P but adenosine did not.

    Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

    Techniques: Ligand Binding Assay, Competitive Binding Assay, Derivative Assay, Binding Assay

    Characterization of ERK1/2 responses to COA-Cl in CHO-EDG1 cells. Shown are the results of protein immunoblot (IB) assay of cell lysates derived from CHO-EDG1 cells and their parental CHO-K1 cells. They were treated with increasing concentrations of COA-Cl (A) and S1P (B). They were then subjected to IB with antibodies directed to phosphorylated and total forms of ERK, as indicated. Left side shows representative images and right side shows graphic summaries, where closed and open circles represent values derived from CHO-K1 and CHO-EDG1 cells, respectively ( n = 4). * P < 0.05 and ** P < 0.01 versus vehicle-treated cells. †† P < 0.01 versus CHO-K1 cells.

    Journal: Pharmacology Research & Perspectives

    Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

    doi: 10.1002/prp2.68

    Figure Lengend Snippet: Characterization of ERK1/2 responses to COA-Cl in CHO-EDG1 cells. Shown are the results of protein immunoblot (IB) assay of cell lysates derived from CHO-EDG1 cells and their parental CHO-K1 cells. They were treated with increasing concentrations of COA-Cl (A) and S1P (B). They were then subjected to IB with antibodies directed to phosphorylated and total forms of ERK, as indicated. Left side shows representative images and right side shows graphic summaries, where closed and open circles represent values derived from CHO-K1 and CHO-EDG1 cells, respectively ( n = 4). * P < 0.05 and ** P < 0.01 versus vehicle-treated cells. †† P < 0.01 versus CHO-K1 cells.

    Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

    Techniques: Western Blot, Derivative Assay

    shCTL or shRGS13 cells were loaded with a calcium-sensing dye, follwed by stimulation with various concentrations of adenosine (A), C5a (B), CXCL12 (C), 100 PM sphingosine 1-phosphate (S-1P) (D) or ionomycin (5 μM, E) and measurement of intracellular Ca2+ concentration by fluorimetry. Each data point represents the mean ± S.E.M. (maximum–minimum value of relative fluorescence units [RFU]) obtained from 3-4 independent experiments (*P=0.004, 1-way ANOVA). Representative kinetic tracings of intracellular Ca2+ after stimulation with each agonist are shown on the right. Time of stimulus addition is indicated by an arrow.

    Journal:

    Article Title: RGS13 controls G-protein-coupled receptor-evoked responses of human mast cells

    doi:

    Figure Lengend Snippet: shCTL or shRGS13 cells were loaded with a calcium-sensing dye, follwed by stimulation with various concentrations of adenosine (A), C5a (B), CXCL12 (C), 100 PM sphingosine 1-phosphate (S-1P) (D) or ionomycin (5 μM, E) and measurement of intracellular Ca2+ concentration by fluorimetry. Each data point represents the mean ± S.E.M. (maximum–minimum value of relative fluorescence units [RFU]) obtained from 3-4 independent experiments (*P=0.004, 1-way ANOVA). Representative kinetic tracings of intracellular Ca2+ after stimulation with each agonist are shown on the right. Time of stimulus addition is indicated by an arrow.

    Article Snippet: Degranulation was triggered in the same buffer with antigen (streptavidin, 1-100 ng/ml) (Sigma) or indicated GPCR agonists (PGE 2 (Calbiochem), 1 μM; adenosine (Sigma), 10 μM; C5a (Sigma), 1 μM; sphingosine-1-phosphate (S-1P) (Sigma), 10-40 μM; complement component C3a (Calbiochem), 5-500 ng/ml) for 30 minutes at 37 °C.

    Techniques: Concentration Assay, Fluorescence