Autoimmune antibodies bound to the RBC membranes.

RBC membranes were prepared as described in “Materials and methods.” Aggregates that formed during the band 3(−/−) mouse membrane preparations were homogenized (see “Materials and methods”). Proteins were separated on 10% SDS-PAGE gels and were immunoblotted with HRP-conjugated, antimouse (A), or antihuman (B) secondary antibody. H and L indicate the positions of the immunoglobulin H and L chains. Mouse RBC membranes are shown in panel A. Loading: C1, C2, controls 1 and 2, respectively; M1, M2, band 3(−/−) mice 1 and 2, respectively; A1, A2, aggregates 1 and 2 from band 3(−/−) mice 1 and 2 preparations, respectively. C1 were C57BL/6 strain; M1, M2, and C2 were B6.129 strain. (B) Human RBC membranes. Loading: C1, C2, controls 1 and 2, respectively. P indicates proband. Molecular weight markers (kDa) are indicated to the left of the blots.