FIG. 3.

Polarized cell surface distribution (A) and TX-100 extraction (B) of chimeric NA TMD proteins. For panel A, confluent monolayers of MDCK cell lines were grown on filters for 3 to 4 days, induced for 16 h with 2 μM CdCl₂, pulse-labeled for 2 h with 300 μCi of [³⁵S]-Easy Tag Express protein-labeling mix and chased for 2 h. The apical (lanes A) and basolateral (lanes B) surface proteins from parallel cultures were biotinylated, isolated by anti-TR antibodies, analyzed by SDS-PAGE, and autoradiographed. For panel B, cells were grown on 35-mm petri dishes for 2 days and induced with 2 μM of CdCl₂ for 16 h, pulse-labeled with 150 μCi of [³⁵S]-Easy Tag Express protein-labeling mix for 2 h, followed by a 2-h chase. Cells were extracted on ice with extraction buffer containing 1% TX-100 for 10 min, as described in Materials and Methods. TX-100 insoluble (lanes I) and soluble (lanes S) proteins were immunoprecipitated with anti-TR antibodies, analyzed by SDS-PAGE, and autoradiographed.