Total protein expression and cell surface fractions are similar for B3, B3SAO, and B3EQ expressed in typical transfected K562 clones.

The same K562 cell samples expressing band 3 mutants that had been used for flow cytometric analysis (Table 1) were used for immunoblotting. The relative amount of band 3 expressed per packed cell volume was determined by scanning densitometry performed on 4 gels, 2 blotted with BRIC 155 (like blot A) and 2 blotted with BRIC 170 (like blot B). The surface fraction of the expressed band 3 proteins was determined by densitometric analysis of 3 gels blotted with BRIC 170 (like blot D) and the mean values of these results are shown in Table 2. Band 3 proteins expressed in K562 cells probably migrated faster than erythrocyte band 3 because of a difference in N-glycosylation. This is consistent with blot C, which shows that the 35-kd chymotrypsin fragment from K562 cells migrated faster than the glycosylated 35-kd fragment from RBCs but slower than the deglycosylated 35-kd chymotrypsin fragment from RBCs. The 63-kd chymotrypsin fragment from the 2 K562/B3EQ clones (and, to a lesser extent, the 2 K562/B3SAO clones) migrated as a double band probably because of a heterogenous posttranslational modification of the expressed protein. These unusual electrophoretic properties had previously been observed for several K562 clones expressing normal band 3.5 MG indicates Memphis I ghosts; −chymo, cells not pretreated with chymotrypsin; +chymo, cells pretreated with chymotrypsin; PNGase F, peptide-N⁴-(N-acetyl-β-glucosaminyl)-asparagine amidase F; pos, positive; neg, negative.