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Figure S1. Pharmacological characterization of HUVEC responses to COA-Cl. (A) Results of cAMP assay. HUVEC were treated for 30 min with forskolin (25 μmol/L) or W146 (10 μmol/L), followed by COA-Cl (100 μmol/L for 10 min), as indicated. They were then subjected to cAMP determination (n = 3). (B) Results of immunoblot (IB) analyses, in which HUVEC were treated with DMS (10 μmol/L for 30 min), an inhibitor of sphingosine kinase, followed by COA-Cl (100 μmol/L for 10 min). Cells were harvested and were subjected to IB using antibodies directed to phospho- and total-ERK1/2, as above (n = 3). (C) Results of receptor desensitization assay. Some cells had been pretreated (“Pre-Tx”) with COA-Cl (100 μmol/L), S1P (100 nmol/L), or vehicle for 60 min prior to COA-Cl (100 μmol/L for 10 min) (n = 3).

Figure S2. Comparison of COA-Cl actions with S1P in HUVEC. (A) Results of immunoblot assays using the cell lysates derived from HUVEC treated with S1P (1 μmol/L) or with COA-Cl (100 μmol/L) for the times indicated, performed in an identical culture batch. They were subjected to IB for phospho- and total-ERK1/2 as above. Blots were also reprobed with antibodies directed to phospho- and total-Akt, as well. The degrees of ERK1/2 phosphorylation were quantified and are summarized in a graph shown in the (B) (n = 4). (C and D) Results of tube formation assays, in which cells were treated with vehicle, VEGF (10 ng/mL), S1P (1 μmol/L), or COA-Cl (100 μmol/L), performed in the identical cellular preparations (n = 5).

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