Figure 3.

Glycosylation and acquisition of detergent insolubility by AE1 anion exchangers occurs after delivery to the plasma membrane. Erythroid cells from a 10-d-old chicken embryo were pulsed for 15 min with ³⁵S-Translabel and chased for 1 h in complete media. The cells were then washed in Ringer’s buffer and incubated in the presence (lanes 1–8) of 1 mg/ml NHS-SS-Biotin in Ringer’s buffer for 30 min at 4^oC. At this time, an aliquot of cells (lanes 1, 2, 3, and 6) was detergent lysed, and AE1 immunoprecipitates were prepared from the detergent-soluble (S) and the detergent-insoluble (I) fractions. One-half of each precipitate was directly analyzed on a 6% SDS polyacrylamide gel (lanes 1 and 2), and the other half was reprecipitated with streptavidin agarose before gel analysis (lanes 3 and 6). The remainder of the cells were chased an additional 1 h (lanes 4 and 7), or 3 h (lanes 5 and 8) in complete media after biotinylation. At the latter time points, AE1 was immunoprecipitated followed by precipitation with streptavidin agarose. Streptavidin-precipitable material was analyzed on a 6% SDS polyacrylamide gel, and labeled AE1 anion exchangers were detected by fluorography. Lanes 9 and 10 are controls that were processed identically to lanes 3 and 6 except the cells were not biotinylated with NHS-SS-Biotin before immunoprecipitation and streptavidin precipitation. Precipitates were digested with neuraminidase before gel analysis.