*2.2. Simultaneous Removal of Long Regulatory Extension (REL) and Helix H9 Significantly Reduces wt-CFTR Processing but Increases Levels of F508del-CFTR Immature Form*

Next, we assessed the impact of removing the long version of RE–RE<sup>L</sup> (∆ <sup>647</sup>Cys-Ser678, Figure 2) which significantly decreased wt-CFTR processing to 88 ± 5% (Figure 2A, lane 3; Figure 2C) but had no impact on F508del-CFTR (Figure 2A, lane 4; Figure 2C). As helix H9 (∆ <sup>637</sup>Gln-Gly646, Figure S1) can be considered to be part of this longer RE region [18] since it interacts with RE (Figure 1A), we also tested the effect of removing helix H9 jointly with ∆REL. Deletion of both H9 (∆H9) and RE<sup>L</sup> further decreased the residual processing of <sup>∆</sup>REL-F508del-CFTR from 9 <sup>±</sup> 2% to 0±0% and in fact the same happened for ∆H9 alone (Figure 2A, lanes 8,6, respectively). Curiously, however, levels of immature F508del-CFTR were significantly increased when both RE<sup>L</sup> and H9 were removed (Figure 2A, lane 8), similarly to ∆RES-F508del-CFTR (Figure 1B) and in contrast to ∆REL-F508del-CFTR.

For wt-CFTR, <sup>∆</sup>REL-∆H9 also led to a decrease in processing (55 <sup>±</sup> 3%), which was more pronounced than for ∆RE<sup>L</sup> alone, but interestingly removal of H9 helix alone only reduced processing to 72 ± 4% (Figure 2A, lanes 7,5, respectively).

Simultaneous removal of RE<sup>L</sup> and helix H9 significantly reduces wt-CFTR processing but increases levels of F508del-CFTR immature form.
