![Fig. 4. Synthesis, stability, and membrane incorporation of GPA in mouse erythroblasts. Band 3 +/+ and band 3 −/− erythroblasts were labeled with [35S]methionine for 60 minutes and then chased with unlabeled methionine for 0, 60, and 120 minutes. Thereafter, the cells were lysed and separated into soluble (ER) and insoluble (membrane) fractions. The identity of the membrane fraction derived from band 3 −/− erythroblasts was confirmed by immunoprecipitation of protein 4.1 (data reviewed, but not shown). Equal volumes of each sample were immunoprecipitated using anti-GPA antibodies and the immunoprecipitates were analyzed by SDS-PAGE. The immunoprecipitation conditions were similar to those described previously.46 The gels were processed for fluorography. The immunoprecipitated bands in the autoradiogram correspond to the position of GPA dimer. The absence of GPA in the membrane fraction of band 3 −/− erythroblasts shows that despite the synthesis of GPA in band 3−/− erythroblasts, GPA is not recruited to the plasma membrane and is presumably degraded rapidly in the cytoplasm. The results of immunoprecipitated GPA were confirmed by three independent experiments. GPa, partially glycosylated glycophorin A; GPA, completely glycosylated glycophorin A.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/91/6/10.1182_blood.v91.6.2146/2/blod4060604.jpeg?Expires=1735830951&Signature=n~uII88~~mTtiu2jMUraGnl4D5Am2M9bxbESVf2I1Mecv3AGZHPgnzdoxrtSa8BlP5uyAJxIDhfVJub8rsmtlIOPY3fv8-bpSQ9~Au8Syu8bqTJFt97DSdOrNyX5u-JaLq~Wu4ZhHqeeJ-ZMGha5p8n1ZHq~OO4GnS4jI6Rw48zLp0AnjitYJmh4aYIbQvsZkaEKT1V7Kevo-OzxcPGhehHXHjUJEY08u9Sqja7qpyeoyvY~KAE~5uYUSXnEPe~8qI6naFka66lLudVikQMV8WHfxpc1WF~ySSAsgvv8GtAI~1wHQeOCJuB5CGRRdrGMEDu9zaA8eaW~t-zdv-DLtA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Synthesis, stability, and membrane incorporation of GPA in mouse erythroblasts. Band 3 +/+ and band 3 −/− erythroblasts were labeled with [35S]methionine for 60 minutes and then chased with unlabeled methionine for 0, 60, and 120 minutes. Thereafter, the cells were lysed and separated into soluble (ER) and insoluble (membrane) fractions. The identity of the membrane fraction derived from band 3 −/− erythroblasts was confirmed by immunoprecipitation of protein 4.1 (data reviewed, but not shown). Equal volumes of each sample were immunoprecipitated using anti-GPA antibodies and the immunoprecipitates were analyzed by SDS-PAGE. The immunoprecipitation conditions were similar to those described previously.46 The gels were processed for fluorography. The immunoprecipitated bands in the autoradiogram correspond to the position of GPA dimer. The absence of GPA in the membrane fraction of band 3 −/− erythroblasts shows that despite the synthesis of GPA in band 3−/− erythroblasts, GPA is not recruited to the plasma membrane and is presumably degraded rapidly in the cytoplasm. The results of immunoprecipitated GPA were confirmed by three independent experiments. GPa, partially glycosylated glycophorin A; GPA, completely glycosylated glycophorin A.
