Figure 2

In vitro characterization of p.Ile1234Val missense mutation introduced into cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA and expressed in heterologous expression system. (a) Immunoblots show the processing of wild-type (WT)-CFTR and p.Ile1234Val-CFTR in human embryonic kidney (HEK) cells after 24 h at 37 °C. WT-CFTR and p.Ile1234Val-CFTR were expressed as both the Golgi-modified, complex glycosylated (mature) band C form (broad 170-kDa band) as well as the endoplasmic reticulum–modified, core glycosylated (immature), band B form (sharp 150-kDa band). Maturation (expressed as percentage of band C/(band B + band C)) of WT-CFTR and p.Ile1234Val-CFTR was quantified for three independent trials, and there was no significant difference between the two (P > 0.05). (b) Fluorescence-based anion flux assay in HEK cells show WT-CFTR and p.Ile1234Val-CFTR function after stimulation using a cAMP agonist (gray bar, forskolin, 10 μmol/l) or vehicle (dimethyl sulfoxide) alone (empty bar). Flux responses in the first 4 min after cAMP stimulation (reported as an initial rate of change in relative fluorescence units (RFU)) for WT-CFTR and p.Ile1234Val-CFTR were quantified for three independent trials (four technical replicates each trial), and there was no significant difference between the two (P > 0.05). Ctl, control; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium.