Band 3 and RhAG extracellular epitopes are disrupted by cell-impermeable pronase treatment. (A) Flow cytometry histograms show the destruction of BRIC6 (band 3) and LA1818 (RhAG) but not BRIC69 (Rh) epitopes on treatment with 500 μg/mL pronase. a.u., arbitrary units. (B) 3 × 10⁷ untreated erythrocytes or erythrocytes treated with 500 μg/mL pronase were lysed and used for each immunoprecipitation with anti–band 3 antibodies BRIC6 and BRAC66. Immunoprecipitates (IPs) were subjected to SDS-PAGE and Western blotting and were immunoblotted with anti–protein 4.2 and anti–band 3 N-terminal-specific antibodies sequentially without stripping. Lane 1 and 2 represent

of the immunoprecipitation input. * indicates original protein 4.2 staining. (C) RhAG (LA1818) and Rh (BRIC69) immunoprecipitations from pronase-treated or untreated intact erythrocytes. Lanes 1 and 2 represent

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of the immunoprecipitation input. Proteins were detected with antibodies raised against the C termini of Rh and RhAG. (D) RhAG immunoprecipitation using a rabbit polyclonal antibody raised against the C terminus of RhAG. Proteins were detected with a carboxyl-terminal–specific Rh antibody or with LA1818 for RhAG. * indicates cross-reactivity with IgG. Erythrocyte lysates represent

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of the immunoprecipitation input.