Coomassie and immunostaining of proteins from Coimbra RBC membranes.

RBC membranes were separated on 10% or 12% Laemmli gels and were immunoblotted using antibodies as shown. Loading: C1, C2, controls 1 and 2, respectively. P indicates proband. (A) Proteins of the band 3 complex. Immunoblotting used polyclonal antibodies against protein 4.2 and monoclonal antibodies: BRIC170 (N-terminal band 3), BRIC155 (C-terminal band 3), and BRIC163 GPA. (B) Proteins of the glycophorin C (GPC) complex, plus aquaporin 1 (AQP1) and cytoskeletal proteins spectrin and actin. Immunoblotting used polyclonal antibodies against protein 4.1, p55, and AQP1 and monoclonal antibodies: BRIC4 (GPC), anti-spectrin, anti–β-actin (Abcam, Cambridge, United Kingdom). (C) Proteins of the Rh complex. Immunoblotting used polyclonal antibodies against the Rh polypeptides and the C-terminal region of CD47, and monoclonal antibodies LA1818 (RhAG), BRIC125 (N-terminal region of CD47), R1.3 (GPB), and BS56 (LW). (D) Other RBC membrane proteins. Immunoblotting used polyclonal antibodies against the glucose transporter (GLUT 1) and monoclonal antibodies BRIC221 (Lu), BRIC128 (DAF), BRIC5 (LFA-3), and BRIC235 (lymphocyte homing receptor [CD44]).