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. Author manuscript; available in PMC: 2019 Apr 8.
Published in final edited form as: Nat Med. 2018 Oct 8;24(11):1732–1742. doi: 10.1038/s41591-018-0200-x

Figure 4.

Figure 4

Corrector combinations rescue the ΔF508-CFTR folding and functional defects. (a) The WT- or ΔF508-CFTR conformation in isolated microsomes was probed by limited trypsinolysis and immunoblotting (left panel). Microsomes isolated from BHK-21 cells, treated with DMSO, VX-809 or 4172+VX-809+3151 corrector combination (3C), were exposed to increasing concentrations of trypsin and the remaining full-length CFTR was quantified by immunoblotting. Complex-glycosylated WT- or ΔF508-CFTR was expressed as the percentage of the initial amount (right panel, n = 4). (b) Correlation between the PM density (n = 3) and band C abundance (n = 5) or activation (Fsk + Gen, n = 3) of ΔF508-CFTR after treatment with single correctors or corrector combinations as determined in Fig. 3c,d and panel (d), respectively. The Pearson correlation coefficient and the associated P-value are shown. (c,d) Effect of indicated single correctors or corrector combinations on the short-circuit current (Isc) of ΔF508-CFTR in CFBE41o-. Representative traces are shown in c. ΔF508-CFTR function (n = 3) normalized with CFTR RNA abundance (Supplementary Fig. 3c) is expressed as percentage of WT function in d. (e,f) The effect of VX-809 or 3C on thermal inactivation of ΔF508-CFTR-2RK reconstituted into an artificial phospholipid bilayer. The Po of protein kinase A–activated CFTRs was analyzed at the indicated temperatures (e), WT (total recording time for each point 16–23 min, n = 11–19, exact number of independent records for each temperature is indicated in the graph), ΔF508–2RK (38–48 min, n = 20–46), ΔF508–2RK + VX809 (6–27 min, n = 10–26), and ΔF508–2RK + 3C (4–18 min, n = 4–16). Significance between the VX-809 and 3C treated channel Po was calculated by two-sided Mann-Whitney U test. *P = 0.04 at 34°C and 0.03 at 36°C. The Po values of untreated ΔF508-CFTR-2RK and WT-CFTR are shown for comparison38. Representative channel activities for ΔF508-CFTR-2RK after VX-809 (two channels) or 3C (single channel) treatment during the temperature ramp are shown in f. The channel open (o) and closed (c) states are indicated. Data in a-b, d and e are means ± SEM of the indicated number of independent experiments.