Figure 2.

Degradation of CFTR and CFTRΔF508 is inhibited by overexpression of UbcH5A C85A. (A) Western blot analysis of CFTR levels upon overexpression of the E2s UbcH5A, Ubc6, and Ubc7. HEK293 cells were transfected transiently with expression plasmids that encode the indicated proteins. Total cell extracts were prepared 24 h after transfection in SDS-PAGE sample buffer. Nitrocellulose was probed with α-CFTR and developed. The immaturely glycosylated ER localized B form and maturely glycosylated plasma membrane associated C form of CFTR are denoted as B and C, respectively. (B and C) UbcH5A C85A overexpression slows the rate of CFTR and CFTRΔF508 degradation. Cells were labeled for 20 min with ³⁵S-translabel and a chase period was initiated by the addition of cycloheximide. At the indicated times, ³⁵S-CFTR or ³⁵S-CFTRΔF508 was immunoprecipitated from cell extracts. CFTR or CFTRΔF508 isolated in this manner was detected by SDS-PAGE and fluorography. (D and E) Graphs illustrate the processing efficiency and half-life of CFTR and CFTRΔF508. Relative CFTR and CFTRΔF508 levels were quantitated by laser densitometry of the x-ray films shown in B and C, respectively. Values were normalized to the quantity of the B form of CFTR and CFTRΔF508 present at t = 0 under the indicated experimental condition levels. t = 0 values for the B form of CFTR were 1.1 and 1.8 OD, in the absence and presence of UbcH5a C85A, respectively. t = 0 values for the B form of CFTRΔF508 were 2.3 and 7.0 OD, in the absence and presence of UbcH5a C85A, respectively. (F) CHIP and UbcH5a jointly reduce levels of CFTRΔF508 in Western blots.