FIGURE 4.

Biotinylation experiments performed to determine the effects of Dab2 and AP-2 on CFTR expression in the plasma membrane of CFBE41o- cells as a function of time. Shown are representative Western blots (IB, A) and a summary of experiments (B) demonstrating that silencing Dab2 attenuated the disappearance of CFTR from the apical plasma membrane. By contrast, silencing μ2 had no effect on the plasma membrane stability of CFTR. The Dab2 p96 was specifically targeted by the siRNA because, unlike p67, Dab2 p96 functions as an endocytic adaptor. Silencing the μ2 adaptin was used to inhibit the AP-2 complex assembly. CFBE41o- cells were transfected with 50 nm siRNA. Disappearance of CFTR from the plasma membrane was monitored over time in the presence of 20 μg/ml cycloheximide (CHX) at 37 °C. In siRNA negative control (siCTRL)-treated cells, ∼50% of CFTR disappeared from the plasma membrane in 24 h. Thus, data are reported at the 24-h time point. Plasma membrane proteins were isolated by selective apical membrane biotinylation. Ezrin expression in the post-nuclear supernatants was used as a loading control (not shown). *, p < 0.05 versus time zero in siCTRL. Three experiments/group were performed. Error bars, S.E.