Figure 2. Influence of palmitoylation on maturation of wild-type CFTR.

(A) Western blot using HeLa cells stably transduced to express wild-type CFTR, CFBE (cystic fibrosis bronchial epithelial cells) stably expressing wild-type CFTR, Calu-3 (airway serous glandular) cells with high level CFTR under regulatory control of the endogenous promoter, and HEK293 cells encoding doxycycline-inducible CFTR in presence or absence of 2-BP (100–150 μM, 8 h). CFTR steady state levels decreased when palmitoylation was inhibited. (B) Pulse-chase analysis of HEK293 cells (expressing wild-type CFTR) labeled with ³⁵S methionine and cysteine followed by a chase in the presence or absence of 150 μM 2-BP. Treatment with 2-BP prevents proper CFTR maturation as shown by failure of band B progression to band C. Results were quantified (C) as a ratio of radiolabeled band C at each time point to starting levels of total (labeled bands B + C) CFTR immediately following the pulse. Halide efflux was measured (D) and quantified (E) by the SPQ fluorescence assay. Forskolin (20 μM) and genistein (50 μM) were added (solid arrow) to stimulate CFTR-dependent ion transport (rate of upward deflection tracks CFTR activity). The findings indicate a decrease in CFTR activity when palmitoylation is inhibited. Dotted arrow = addition of dequenching buffer; double arrow = addition of quenching buffer. Results are shown as mean ± SEM and normalized to control cells. *P < 0.05, **P < 0.005; n = 4.