Fig. 4. ER retention does not destabilize wild-type Shaker protein. (A) Shaker-expressing cells were treated with brefeldin A (5 µg/ml) and nocodacole (20 µg/ml) (BFA/NOC) or were untreated. Cells were metabolically labeled for 10 minutes and were harvested immediately (0 h) or after chase periods of 24 or 48 hours, as indicated. Open and closed arrowheads indicate the positions of the immature and mature forms of the Shaker protein, respectively. Compiled data from 5-8 similar experiments indicated that the amount of Shaker protein remaining at 48 hours was 94±12% in untreated cells and 116±22% in BFA/NOC-treated cells. (B,C) Representative differential interference contrast (DIC) (i) and confocal images (ii-iv) of HEK293T cells transiently transfected with wild-type Shaker are shown. At 24 hours post-transfection, cells were incubated in the presence of BFA/NOC (B) or left untreated (C), before permeabilization, labeling with antibodies against calnexin (ii) and Shaker (iii), and visualization with fluorescently conjugated secondary antibodies. Red, calnexin; green, Shaker. The merged images are shown in iv. Bars, 10 µm.