Western blot analysis of the red blood cell membrane proteins. Red blood cells were collected from both band 3 +/+ and band 3 −/−mice and washed in PBS. Ghosts were prepared by lysis as described in Materials and Methods. Ghosts membranes were solubilized and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to nitrocellulose, Western blot analysis was performed using the ECL chemiluminescence kit as suggested by the manufacturer. Both band 3 +/+ and band 3 −/− red blood cell membranes contain protein 4.1, glycophorin C, p55, dematin, and adducin, while the complete absence of protein 4.2 and GPA is noted. The GPA panel shows the position of the dimer. No monomeric GPA was detected (data reviewed, but not shown). It should be noted that each membrane fraction used for Western blotting was isolated from the same number of normal and band 3 −/− red blood cells. However, a more precise quantitative method such as enzyme-linked immunosorbent assay (ELISA) will be required to compare the absolute amounts of membrane proteins in normal and band 3 null red blood cells.