Fig. 8.

The impact of CFTR modulators on the accumulation of short CFTR fragments that contain MSD1 and different lengths of C-terminal extensions. (A) Schematic diagram of CFTR fragments whose accumulation becomes sensitive to modulators upon inclusion of residues that are located between amino acid 375 and 380. (B) Comparison of ABBV-2222, lumacaftor, and tezacaftor on the steady-state accumulation of the indicated CFTR fragment. Indicated modulators were added at 5 µM to cultures of human embryonic kidney (HEK) 293 cells 14 hours prior to harvest and detection of the fragment. Modulators were added to cells that expressed 380X-CFTR at T = 0 of the chase reaction in the same media with the protein synthesis inhibitor cycloheximide (10 µg/ml). The pCDNA3.1 expression plasmid (0.25 µg) that harbored the indicated form of CFTR was transfected into HEK293 cells at 18 hours prior to harvest. CFTR fragment expression was detected by western blot with the CFTR N-terminal tail antibody (MM13-4). In (B), the level of respective CFTR fragment was quantitated with a GE Healthcare LAS 4000 and compared with levels detected in the presence of the proteasome inhibitor bortezomib (10 µM), which was added to cultures 4 hours prior to harvest. This was done because CFTR fragments 370X, 373X, and 375X have a short half-life and do not accumulate to detectable levels unless they are stabilized by folding modulators or inhibition of the proteasome. In (C), changes in CFTR signals over the course of a 2-hour chase reaction were normalized to the CFTR signal at T = 0.