Analysis of the effect of o-vanadate treatment on erythrocyte morphology and vesicle release. (A) Images of 2mM o-vanadate–treated (o-v) erythrocytes at different incubation times. (B) Confocal fluorescence microscopy images (maximum intensity projections) of DiI-labeled untreated erythrocytes (panel 1) and 2mM o-vanadate–treated erythrocytes (panel 2), both at 60 minutes. (C) Confocal microscopy images of erythrocytes treated with 2mM o-vanadate for 1 hour, erythrocytes labeled with anti–band 3 (panel 1) and anti-phosphotyrosine (anti-pTyr; panel 2) antibodies, and an overlay image (panel 3). (D) Electron microscopy image of the vesicles released from o-vanadate–treated erythrocytes. Ultrathin sections were stained with osmium tetroxide, examined, and photographed using a Zeiss EM 900 transmission electron microscope operating at 80 KV. Bar indicates 1 μm. Vesicles released from erythrocytes treated with increasing o-vanadate (o-v) concentrations (E) or with 2mM o-vanadate for different incubation times (F) were collected by ultracentrifugation from the supernatants of cells incubated at 42°C for 45 minutes. The amount of released vesicles was estimated by measuring band 3 using anti–band 3 Western blotting. Vesicle proteins obtained from o-vanadate–treated erythrocytes were separated by 8% SDS-PAGE under reducing conditions. Gels were stained with Coomassie (lane 1) or transferred to nitrocellulose and immunostained with anti–band 3 (lane 2), anti-adducin (lane 3), and anti-spectrin (lane 4) antibodies (G). Images were acquired using a laser infrared fluorescence detector (Odyssey; LI-COR Biosciences).