Figure 5.

Fate of newly synthesized AE1 after removal of ammonium chloride from the medium. Erythroid cells from a 10-d-old embryo were pulsed for 15 min and chased for 45 min in complete media. At this time, 25 mM ammonium chloride was added to the media of some of the cells, and the cells were incubated until a total of 4 h of chase had elapsed. Treated cells (lanes 3 and 4) and untreated control cells (lanes 1 and 2) were then detergent lysed and separated into soluble (S) and insoluble (I) fractions, and AE1 immunoprecipitates were prepared from each fraction. After the 4-h chase period in 25 mM ammonium chloride, aliquots of cells were also pelleted and resuspended in complete media lacking the reagent. The cells were then incubated an additional 10 min (lanes 5 and 6), or 30 min (lanes 7 and 8) at 37°C in reagent-free media and processed for immunoprecipitation using AE1-specific antibodies. Immunoprecipitates were digested with endo F before analysis on a 6% SDS polyacrylamide gel.