Figure 2. Lipid rafts participation in H₂O₂-induced Akt prosurvival signaling. A) BAEC were pretreated with Wortmannin (2 µM) for 1 h followed by bolus administration of H₂O₂ for times indicated. Inhibition of PI3 kinase (Wort) showed enhanced H₂O₂-induced caspase 3 cleavage. B) The number of cells containing fragmented DNA (TUNEL assay) was significantly greater in wortmannin-treated monolayers subjected to H₂O₂ (4 h) compared to H₂O₂ alone. C) Control and CD-pretreated BAEC were exposed to H₂O₂ for 15 min and then processed to purify plasma membranes. The plasma membranes were sonicated and subfractionated by sucrose gradient (5% to 30%) centrifugation to isolate light buoyant density lipid rafts domains from bulk membranes. Plasma membrane fractions were Western-blotted with indicated primary antibodies. (D) H₂O₂ enhanced phosphorylation of Akt over nontreated control cells. Disassembling rafts with CD significantly attenuated H₂O₂-induced Akt phosphorylation. Data are reported as mean ± SD; *P < 0.05. Comparisons between H₂O₂-treated cells with and without CD were significant at P < 0.05 (#).