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. Author manuscript; available in PMC: 2011 Aug 29.
Published in final edited form as: Circ Res. 2009 Mar 12;104(8):978–986. doi: 10.1161/CIRCRESAHA.108.193367

Figure 5.

Figure 5

OxPAPC and S1P1 receptor regulate Akt phosphorylation and human EC barrier enhancement. A, ECs were either treated with scrambled siRNA or S1P1 receptor siRNA,7 grown to confluence, serum-starved for 1 hour, and either left untreated (control) or treated with 20 μg/mL OxPAPC for 5 minutes. ECs were then solubilized and analyzed using immunoblotting with anti–phospho-tyrosine (Y326) Akt (a), anti–phospho-serine (S473) Akt (b), anti–phospho-threonine (T308) Akt (c), or anti-Akt (d) antibody. B, Graphic representation of the ratio of tyrosine (Y326) phosphorylated Akt, serine phosphorylated (S473), or threonine phosphorylated (T308) to total Akt in EC lysates treated with 20 μg/mL OxPAPC (5 minutes) with or without scrambled siRNA, S1P1 receptor siRNA 1,13 S1P1 receptor siRNA 2 (Santa Cruz Biotechnology), S1P1 receptor antagonist W146 (250 nmol/L, 1 hour), mTOR siRNA, or PI3-kinase inhibitor LY294002 (10 μmol/L, 1 hour). C, ECs were plated on gold microelectrodes, treated with scrambled siRNA, or Akt1 siRNA,13 grown to confluence, serum-starved for 1 hour, and either left untreated (control) or treated with 20 μg/mL OxPAPC. The arrow indicates the time of OxPAPC addition. D, ECs were plated on gold microelectrodes, treated with scrambled siRNA, or S1P1 receptor siRNA,13 grown to confluence, serum-starved for 1 hour, and either left untreated (control) or treated with 20 μg/mL OxPAPC. The arrow indicates the time of OxPAPC addition. E, Graphic representation of the percentage maximal OxPAPC-induced transendothelial cell electric resistance (y axis) using siRNA and inhibitors (x axis) as we have described in Figure 3B and Figure 5B through 5D.