Immunoblotting analysis of RBC membrane glycophorins. (A) Immunoblots probed with monoclonal anti-He. Lane designations are: 1, He−, He negative control; 2, He+, He positive control; and 3, proband GL. He monomer is seen in He+ but not He−, whereas two monomeric components of GPHe are present in GL (indicated by He and an arrow). Note that the upper bands seen in lane GL are due to overload and formation of homodimers and heterodimers with other glycophorins. (B) Immunoblots probed with monoclonal anti-N. Note that RBC membranes from proband GL contain a GPB form comparable to that from He− control (B denotes the monomeric form). (C) RBCs were treated with α-chymotrypsin and then membranes were prepared for immunoblotting with anti-He. A minus sign (−): untreated, and a plus sign (+): chymotrypsin-treated. This enzyme digestion almost completely removed the GPHe monomer with a size comparable to GPB but not at all the lower-molecular-weight GPHe species (arrow-indicated).