The pronase-resistant internal pool of band 3 in basophilic erythroblasts is EndoH sensitive. (A) 1.5 × 10⁷ cells differentiated for 48 hours were either left untreated (lanes 1 and 5) or were treated with pronase only (lanes 2 and 6), BFA + pronase (lanes 3 and 7), or CHX + pronase (lanes 4 and 8) as described in “Deglycosylation of immunoprecipitates.” Cells were then lysed and used to immunoprecipitate band 3 using BRIC6 (lanes 5-8). Immunoprecipitated proteins were detected by immunoblotting with polyclonal antibodies raised against protein 4.2 and the C terminus of band 3 sequentially without stripping. Lanes 1-4 represent the total cell lysate of

of the immunoprecipitation input. (B) Cells differentiated for 48 hours were left untreated or were pretreated with 100 ng/mL CHX or the indicated concentrations of BFA for 3 hours followed by 500 μg/mL pronase treatment. Reappearance of the BRIC6 (band 3) epitope at the cell surface was monitored at the indicated time points by flow cytometry. Data are expressed as mean fluorescence intensity (arbitrary units [a.u.]); n = 2 for each treatment. (C-D) Cells removed from culture after 48 hours of differentiation were treated or not with 500 μg/mL pronase and lysed. (C) BRIC6 was used to immunoprecipitate the internal pool of band 3. Immunoprecipitates were treated with EndoH, PNGase F, or water as a control as detailed in “Immunoprecipitates and total cell lysates,” and proteins were separated on 6% gels. Blots were stained with anti–band 3 carboxyl-terminal antibody. Note that the pronase-insensitive BRIC6 immunoprecipitated band 3 is sensitive to both EndoH and PNGase F. (D) Total cell lysates from the pronase-untreated sample were treated with EndoH, PNGase F, or water as a control and proteins were separated by SDS-PAGE. Blots were stained with anti–band 3 carboxyl-terminal antibody. Note that the majority of band 3 is sensitive to PNGase F but not to EndoH.