SDS-PAGE and immunoblotting of band 3 Walton.

(A-D) Membranes prepared and separated by SDS-PAGE. (A) Membranes from chymotrypsin-treated red cells stained for protein with Coomassie blue. (B) Membranes from band 6–depleted red cells2immunoblotted using sheep antihuman carbonic anhydrase II (Serotec, Oxford, England). The variation in intensity of the band reflects the different amounts of total protein loaded on each track. The total protein in each track was estimated from scans of the total spectrin bands in parallel gels stained with Coomassie blue. (C) Fluorograph of membranes from chymotrypsin-treated red cells labeled with 2 μM [³H]H₂DIDS. (D) Membranes from chymotrypsin-treated red cells were digested with peptide N-glycosidase and immunoblotted using monoclonal antibodies. For panels A-D, track 1 represents control membranes; track 2, membranes from affected individual B1; track 3, membranes from affected individual B2. Arrows in panels A and C indicate the normal (60 kd) and band 3 Memphis (63 kd) NH₂-terminal chymotryptic fragments, and arrows in panel D indicate the normal and band 3 Walton COOH-terminal chymotryptic fragments. (E) Location of monoclonal antibody epitopes in the C-terminal portion of normal band 3. Only the region of band 3 containing membrane spans 12 to 14 is shown. BRIC155 and BRIC130 are directed against the COOH-terminal cytoplasmic tail of band 3, while BRIC132 is directed against the final intracellular loop of band 3.