CFTR Maturation Efficiency Assay

Experiments were performed essentially as described^{9 (link)}. Briefly, 24 hours after start of compound treatment, CFBE41o- cells expressing WT- or ΔF508-CFTR were pulse-labeled with 0.2 mCi/ml ³⁵S-methinonine and ³⁵S-cysteine (EasyTag Express Protein Labeling Mix, PerkinElmer) in cysteine and methionine-free medium for 20 minutes at 37°C or labeled for 40 minutes and then chased 2.5 hours at 37°C in full medium. Radioactivity incorporated into the core- and complex-glycosylated glycoproteins was visualized by fluorography and quantified by phosphoimage analysis using a Typhoon imaging platform (GE Healthcare). The maturation efficiency was determined by calculating the percent of pulse-labeled immature, core-glycosylated CFTR conversion into the mature, complex-glycosylated form after 2.5 hours of chase.