Pulse-chase analysis of CFTR variant trafficking

Experiments were performed as described (11 (link)). Briefly, CFBE cells expressing ΔF508-CFTR were pretreated with 3 µM VX-809 for 24 hours followed by 1-hour exposure to 1 or 0.1 µM VX-770. CFBE cells expressing ΔF508-CFTR-3S were exposed to 1 µM VX-770 for 1 hour. CFTR variants were pulse-labeled with [³⁵S]methionine and [³⁵S]cysteine (0.2 mCi/ml) (EasyTag EXPRESS Protein Labeling Mix, PerkinElmer) in cysteine- and methionine-free medium for 30 min and chased in full medium for 2.5 or 4.5 hours at 37°C in the presence of the indicated compounds. Radioactivity incorporated into the core- and complex-glycosylated CFTR was visualized by fluorography and quantified by phosphorimage analysis with a Typhoon imaging platform (GE Healthcare).