Figure 7.

Acquisition and maintenance of detergent insolubility by AE1-4. MDCK cells stably expressing the AE1-4 anion exchanger were pulsed with ³⁵S-Translabel™ for 15 min, and chased for times ranging from 0–48 h. At each time point, cells were detergent lysed and separated into soluble and insoluble fractions by centrifugation. Immunoprecipitates were prepared from each fraction using antibodies generated against the cytoplasmic domain of AE1-4. Immune complexes were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography (A). Confluent or 50% confluent monolayers of MDCK cells expressing AE1-4 were incubated in the absence or presence of 25 μg/ml latrunculin B for 1 h. Cells were then detergent lysed, and the detergent soluble and insoluble fractions were analyzed by immunoblotting analysis using AE1-specific peptide antibodies (B).