Zeta potential (ZP) analyses.

<p>(A) Diagram of ZP principle. ZP is defined as the electrochemical potential at the shear plane. Outside the shear plane, ions are not closely associated with the internal ion cloud. (B) ZP measurements from AA and CC erythrocyte populations. Peak values were estimated by Gaussian fitting the histogram. (C) Levels of membrane-associated hemichromes (mean±SD) in control and NaNO₂-treated AA erythrocytes. For reference, native CC erythrocytes show 1.8-fold greater hemichrome levels than native AA erythrocytes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005828#pone.0005828-Fairhurst1" target="_blank">[31]</a>. (D) ZP measurements from control and NaNO₂-treated AA erythrocyte populations.</p

Detergent-resistant membrane analyses of AA and CC erythrocytes.

<p>Proteins from 18 fractions (out of a total of 36 fractions) were separated by SDS-PAGE under denaturing conditions, transferred to PVDF membrane, and probed with protein-specific monoclonal antibodies.</p

Flow cytometry analyses of phosphatidylserine on the surface of unfixed AA and CC erythrocytes.

<p>Erythrocytes were reacted with FITC-labeled Annexin-V. Subpopulations of AA (A) and CC (B) erythrocytes were gated and the percentage of total erythrocytes within each gate was calculated. Normalized mean fluorescence intensities of the low and high side scatter populations (black and red arrows, respectively) are shown in panel C. Error bars represent SEM (n = 4).</p

Distributions of relative band 3 and CD47 signals from dot blot analyses.

<p>For each protein, the sum of signals from all fractions was set as 100%. Band 3 (A, B) and CD47 (C, D) signals showed similar distributions in the high density fractions of AA and CC samples. However, differences in band 3 and CD47 signal distributions were observed in the low density fractions of AA and CC samples. Higher density signals for both proteins in CC samples appeared in fractions 9–11, which correspond to visible DRM.</p

Analysis of membrane-associated IgG in AA and CC erythrocytes.

<p>Proteins from 9 fractions (out of a total of 36 fractions) were separated by SDS-PAGE under denaturing conditions, transferred to PVDF membrane, and probed with a monoclonal antibody specific for human IgG.</p

Lipid composition of AA and CC erythrocytes.

<p>All mean values are shown as mole % of total lipids including cholesterol. Error bars represent SEM (n = 3).</p