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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 1.

Figure 1

CEMs regulate OxPAPC-mediated human EC barrier enhancement. A, ECs were grown to confluence, serum-starved for 1 hour, and either left untreated (control) or treated with 20 μg/mL OxPAPC for 5, 15, 30, 45, or 60 minutes. Cellular material was analyzed using immunoblotting with anti–phospho-tyrosine–caveolin-1 (a) or anti–caveolin-1 (b) antibody. B, ECs were plated on gold microelectrodes, serum-starved for 1 hour, and treated with either PBS (pH 7.4) (control) or 5 mmol/L MβCD (a cholesterol-depletion agent that abolishes CEM formation) 30 minutes before PBS (pH 7.4) or 20 μg/mL OxPAPC addition. The arrows indicate the times of MβCD and OxPAPC addition. C, ECs were grown to confluence, serum-starved for 1 hour, and either left untreated (control) or treated with 5 mmol/L MβCD 30 minutes before PBS (pH 7.4) or 20 μg/mL OxPAPC addition (5, 15, or 30 minutes). ECs were then solubilized and incubated with p21-binding domain (PBD)-conjugated beads to bind activated (GTP-bound form) Rac1. The PBD bead-associated material was analyzed using immunoblotting with anti-Rac1 antibody. D, ECs were grown to confluence, serum-starved for 1 hour, and either left untreated (control) or treated with 5 mmol/L MβCD 30 minutes before PBS (pH 7.4) or 20 μg/mL OxPAPC addition (5, 15, or 30 minutes). ECs were then solubilized and analyzed using immunoblotting with anti–phospho-tyrosine–caveolin-1 (a), anti–phospho-PAK1 (b), or anti–phospho-tubulin antibody (c). E, ECs were grown to confluence on glass coverslips, serum-starved for 1 hour, and either left untreated (control) or treated with 5 mmol/L MβCD 30 minutes before PBS (pH 7.4) or 20 μg/mL OxPAPC addition (15 minutes). Cells were then fixed and stained with TRITC-phalloidin (to visualize F-actin) and analyzed using fluorescent microscopy.