RLIP76 expression and localization.(A) EAhy926 were immunoprecipitated with mouse polyclonal anti-RLIP76 C-ter antibodies. The immunoprecipitates were analyzed by Western blotting, using anti–human RLIP76 C-ter antibodies. Bound antibodies were visualized with HRP-conjugated anti–human IgG and immunoreactivity was assessed by ECL. Virtually no reactivity was found with immunoprecipitates obtained using non RLIP76-specific IgG (irrelevant). (B) RLIP76 expression in vascular tissue was detected by immunohistochemistry on tissue arrays with histologic sections from normal vascular human tissue incubated with mouse anti-RLIP76 C-ter antibodies. Intense immunoreactivity was observed in vascular endothelium. (C) Immunofluorescence analysis of RLIP76 distribution in EAhy926 untreated cells (left panel) or treated with 30 μM H₂O₂ 30 minutes (right panel). Original magnification 500×, objective 100×, numeric aperture 1.4. (D,E) Flow cytometric analysis after surface staining of EAhy926 cells with antibodies to the RLIP76 C-terminus. (D) Results obtained in a representative experiment. Full light-gray histogram: untreated control cells; red histogram: H₂O₂-treated cells incubated for 30 minutes with fresh medium; black histogram: H₂O₂-treated cells incubated for 6 hours with fresh medium; blue histogram: H₂O₂-treated cells incubated for 24 hours with fresh medium. (E) Time-course evaluation of RLIP76 expression in untreated cells and in cells treated with H₂O₂ and then incubated for 30 minutes, 6 hours, and 24 hours in fresh medium (mean ± SD of the results obtained from 3 different experiments). *P < .01 by Student t test.