Immunoblot analysis of RBC membrane proteins. Total membrane proteins (60 μg) separated on SDS-PAGE were blotted on nitrocellulose sheets and incubated with the murine anti-Fy6 MoAb i3A and with a rabbit anti-p55 antibody. The DARC glycoproteins and p55 proteins were visualized by chemioluminescence autoradiography using goat antimouse or goat antirabbit IgG conjugated to horseradish peroxidase, as second antibodies, respectively. The 35- to 45-kD DARC glycoprotein forms aggregates migrating as bands of 90 and 200 kD.45