Fig. 1. Analysis of band 3 clustering in uninfected and parasitized erythrocytes. (A) Immunoblot of membrane extracts from both uninfected and parasitized erythrocytes using a mAb against the band 3 cytoplasmic domain (cdb3). All lanes, except for parasitized CC erythrocytes, were loaded with 0.1 µg of total protein. 0.3 µg of total protein from parasitized CC erythrocytes was used to obtain approximately the same amount of signal from band 3 protein. The arrows point out the signal from band 3 oligomers found in parasitized AA and CC erythrocytes. Much greater oligomerization is apparent in the lane of parasitized CC erythrocytes. (B) Quantification of band 3 in SE-HPLC fractions of AA and CC erythrocyte membrane extracts. The amount of protein used in CC extracts (0.63 µg/µl) was approximately double that in AA extracts (0.33 µg/µl). Band 3 in the SE-HPLC fractions was semi-quantified by ELISA using mAb against cdb3 at 1/5000 and 1/2000 dilutions. The anti-band 3 signal obtained for AA extracts is markedly higher than for CC extracts. In a competition ELISA, increasing the amount of mAb caused only a minor increase in anti-band 3 signals.