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. 2022 Dec 16;11(24):4096. doi: 10.3390/cells11244096

Figure 2.

Figure 2

The complex allele [G576V;R668C] increases the activation and maturation of the CFTR channel. The functional and biochemical analysis of the [G576A;R668C] CFTR complex allele on heterologous expression systems. (A). Increase in CFTR activation. The bar graphs show the activity of [G576A;R668C] CFTR and, for comparison, WT-CFTR transiently expressed in CFBE41o- (upper panel) or FRT (lower panel) cells stably expressing HS-YFP. The CFTR activity was determined as a function of the YFP quenching rate following the iodide influx in cells stimulated with FSK alone (20 µM; light gray) or with FSK plus ivacaftor (1 µM; dark gray). The data are means ± SD (n = 3). The asterisks indicate statistical significance vs. the WT-CFTR protein: **, p < 0.01. (B). Increase in CFTR maturation. Representative Western blot images showing the electrophoretic mobility of [G576A;R668C] and, for comparison, wild-type, G576A, R668C, and F508del CFTR transiently expressed in CFBE41o- (upper panel) or FRT (lower panel) cells. The whole lysates derived from cells not expressing CFTR (null cells) are shown as controls for antibody specificity. In the case of F508del, the cells were treated for 24 h with DMSO alone (vehicle) or Elexa/Teza (3 µM/10 µM) to correct the mutant misfolding. The arrows indicate the complex-glycosylated (band C) and core-glycosylated (band B) forms of the CFTR protein. (C). CFTR band C densitometry of the Western blot experiments. The data are means ± SD (n = 3). The asterisks indicate statistical significance vs. WT-CFTR protein: **, p < 0.01. (D). CFTR half-life evaluation. Representative Western blot image showing the CFTR expression pattern of [G576A;R668C] CFTR and, for comparison, WT-CFTR transiently expressed in CFBE41o- cells at different time points (T = 0 and 24 h) following the CHX-induced block of protein synthesis. The whole lysates derived from cells not expressing CFTR (null cells) are shown as controls for antibody specificity. (E). The quantification of the normalized CFTR band C (left graph) following CHX treatment (T = 0 h, light gray; T = 24 h, dark gray) and band C fold-increase (right graph) over 24 h CHX treatment, obtained from experiments detailed in D, normalized with the initial value of band C. The data are means ± SD (n = 3). The asterisks indicate statistical significance vs. WT-CFTR protein: **, p < 0.01.