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
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![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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6426/pmc04186426/pmc04186426__prp20002-e00068-f10.jpg)
