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Fig. 7. Transient interaction with calnexin provides long-term protection from ERAD. (A) Shaker-expressing cells were treated with BFA/NOC or were untreated, as indicated. Cells were metabolically labeled for 2 minutes, chased for the indicated times and lysed. Proteins were precipitated sequentially with antibodies directed against calnexin and Shaker (Nagaya et al., 1999). A representative experiment is shown; n=3. Open arrowhead indicates the position of the core-glycosylated, immature form of the Shaker protein. (B) The amount of Shaker protein obtained after sequential immunoprecipitation with calnexin and Shaker antibodies in the presence (
) or absence (
) of BFA/NOC treatment was quantified by densitometry, normalized to the maximum value obtained during the experiment and plotted versus chase time. A representative experiment is shown; n=3. (C) Comparison of the time course of calnexin interaction and ERAD for the wild-type Shaker protein. The percentage of Shaker protein remaining at 0, 24 and 48 hours of chase after treatment with BFA/NOC (
) or with BFA/NOC and dNJ (
) was quantified by densitometry and plotted versus time of chase. The values of the percentage of Shaker protein remaining in BFA/NOC-treated samples were 160±44% (24 hours) and 83±4.9% (48 hours) and in BFA/NOC+dNJ-treated samples were 83±8% (24 hours) and 31±5% (48 hours). 
P<0.001 for BFA/NOC/dNJ versus BFA/NOC at the 48 hour chase time; Student's t-test. The data from B have been replotted on the same axes using the same symbols as in B (, ).
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