Band 3 and protein 4.2 interact initially in an intracellular compartment. (A) Example of flow cytometry histograms confirming destruction of BRIC6 (band 3) and LA1818 (RhAG) epitopes on differentiating erythroblasts at 24, 72, and 144 hours of differentiation. (B) 1 × 106 cells from indicated stages of differentiation were either treated (+) or not (−) with 500 μg/mL pronase before lysis (T = time in hours). Proteins were immunoblotted with the N-terminal intracellular epitope antibody BRIC170 (band 3). Intact uncleaved band 3 and N-terminal cleavage products are indicated. (C) Total cell lysate from equal numbers (1 × 106) of a purified reticulocyte population (RETICS) or erythrocytes (RBCS) treated or not with 500 μg/mL pronase were immunoblotted with anti–band 3 antibody BRIC170. Note the existence of a pool of internal band 3 (∼ 1% by densitometry) in pronase-treated reticulocytes that is absent in pronase-treated erythrocytes. (D) 1.5 × 107 cells at the indicated stages of differentiation were used for total (T), internal (I), or surface (S) immunoprecipitations, respectively, using the band 3 antibody BRIC6 as described in “Immunoprecipitations and total cell lysates.” Proteins were detected by immunoblotting with polyclonal antibodies raised against protein 4.2 and the C terminus of band 3 sequentially without stripping. * indicates the original protein 4.2 staining before detection of band 3.
