Membrane skeleton proteins in wan homozygotes. (A-E) Western blots showing levels of spectrin (sp, A), protein 4.1 (4.1, B), ankyrin (ank, C), band 3 (bd 3, D), and protein 4.2 (4.2, E) in normal (+/+) and wan/wan (-/-) RBC ghosts. Note the absence of band 3 and protein 4.2 and the decrement of ankyrin in mutant RBCs. In panel D, a peptide antibody raised to amino acids 214 to 228 of the cytoplasmic domain of band 3 was used. The same result was obtained using a pan-cytoplasmic domain band-3 antibody as well as a peptide antibody recognizing the C-terminal 12 amino acids of the membrane spanning domain, confirming that wan homozygotes are band-3 null. (F) Northern blot showing the severe decrement in band-3 mRNA in wan homozygotes. Each lane contains 2 μg total RNA purified from 2 pooled mutant (-/-) and 4 phenotypically normal (+/?) newborn peripheral blood samples whose genotypes were subsequently confirmed by PCR (see “Materials and methods”).