FIGURE 7:

Relative stoichiometry of Hsp/c70 and CBag determines CFTR stability. (A) Wild-type or ΔF508 CFTR was cotransfected with CBag or Bag-1 into HEK293 cells. Forty-eight hours after transfection, cells were harvested, and lysates were analyzed by immunoblotting with anti-CFTR antibody. Immature ER (Band B) and Golgi-processed (Band C) forms are indicated. (B) CFTR levels were quantitated by densitometry and are presented as mean ± SEM (n = 5). (C and D) Wild type (C) and ΔF508 (D) CFTR were transfected +/– CBag in HEK293 cells. Twenty-four hours after transfection, cells were pulsed with [³⁵S]Met/Cys for 15 min, and chased for indicated times as described in Materials and Methods. Whole-cell lysate was immunoprecipitated with anti-CFTR antisera and analyzed by SDS–PAGE and phosphorimaging. Asterisk denotes a background band. (E) Immunnoblot of Hsc70, Hsp70, Bag-1, and CBag in whole-cell lysate (25 μg) from transfected cells as in (A). Purified His-tagged proteins used to quantitate results are shown in the right three lanes of each gel. Ratio of total endogenous Hsp/c70 to transfected CBag and Bag-1 are estimated to be 1:0.04 and 1:0.13, respectively (see Table 1).