Fig. 6. Shaker wild-type protein remains competent for ER-to-Golgi transport despite treatment with BFA/NOC and dNJ. (A) Cells expressing the Shaker wild-type protein were pre-incubated with BFA/NOC and dNJ for 2 hours; the drugs were then maintained during a 30 minute starvation period, 30 minute pulse and 2 hour chase. After 2 hours of chase, cells were washed with PBS and the medium was replaced. The chase was then continued in the presence of dNJ with or without BFA/NOC. At various times, cells were harvested and solubilized with detergent, and the Shaker protein was immunoprecipitated and subjected to electrophoresis and fluorography. Lanes show Shaker protein from cells: (1) harvested immediately after pulse; (2) harvested after 2 hours of chase; (3) harvested after 9 hours of chase in the presence of dNJ and BFA/NOC; (4) harvested after 9 hours of chase in the presence of dNJ but no BFA/NOC after the first 2 hours of chase; (5) untreated with dNJ and BFA/NOC and harvested after 9 hours of chase. Open arrowhead indicates the position of the immature form of the Shaker protein. Closed arrowhead indicates the position of the mature form of the Shaker protein from untreated cells (lane 5). Filled circle indicates the position of a diffuse band seen upon removal of BFA/NOC (lane 4). This putative mature band was first detected approximately 2-3 hours after the removal of BFA/NOC. Representative results are shown from a total of six experiments. (B) Cells expressing wild-type Shaker were preincubated, starved, pulsed and chased for 2 hours in the presence of dNJ and BFA/NOC. At 2 hours of chase, cells were washed with PBS and the medium was replaced. The chase was then continued for 3 more hours in the presence of dNJ with (lane 1) or without (lanes 2-4) BFA/NOC. Lane 3: immunoprecipitated protein was treated with endo H. Lane 4: immunoprecipitated protein was treated with PNGase F. Filled circle indicates the position of the diffuse band seen upon removal of BFA/NOC. Open arrowhead indicates the position of the immature form of the Shaker protein. Open square indicates the deglycosylated Shaker protein. Representative results are shown from a total of two experiments. (C) Cells expressing the misfolded Shaker mutant D316K were preincubated, starved, pulsed and chased for 2 hours in the presence of dNJ and BFA/NOC. The chase was then continued in the presence of dNJ with or without BFA/NOC. Lanes show D316K protein from cells: (1) harvested after 2 hours of chase; (2) harvested after 6 hours of chase in presence of dNJ and BFA/NOC; (3) harvested after 6 hours of chase in presence of dNJ but no BFA/NOC after the first 2 hours of chase; (4) untreated with dNJ and BFA/NOC and harvested after 6 hours of chase. Open arrowhead indicates the position of the immature form of the Shaker protein. Representative results are shown from a total of two experiments.