*2.5.* ∆*RIL-*∆*RES-F508del-CFTR Levels at the Plasma Membrane are Equivalent to Those of wt-CFTR*

To determine the fraction of the above CFTR variants that localize to the PM, we used quantitative cell surface biotinylation. These data showed that PM levels of ∆RIL-∆RES-F508del-CFTR were equivalent to those of wt-CFTR, while those of ∆RIL-F508del-CFTR were significantly lower (Figure 4A, lanes 5,4; Figure 4B). Data also confirmed that ∆RE<sup>S</sup> did not induce appearance of F508del-CFTR at the cell surface (data not shown). Corrector VX-809 further increased the PM expression of ∆RIL-∆RES-F508del-CFTR to levels that are significantly higher than those of wt-CFTR (Figure 4A, lanes 2,9, Figure 4B). This compound also significantly increased PM levels of ∆RIL-F508del-CFTR to similar levels of wt-CFTR (Figure 4A, lanes 2,8; Figure 4B).

**Figure 4.** Plasma membrane levels, efficiency of processing and turnover of immature CFTR without Regulatory Extension (RES). (**A**) BHK cells expressing ∆RIL-F508del and ∆RIL-∆RES-F508del-CFTR and treated with 3 µM VX-809 for 48 h (or DMSO control) were subjected to cell surface biotinylation. wt-CFTR samples not treated with biotin were the negative control (NC). After streptavidin pull-down, CFTR was detected by WB. CFTR and CNX are detected in the whole cell lysate (WCL) as controls. (**B**) Quantification of data in (a) for PM CFTR normalized to total protein and shown as fold change relatively to wt-CFTR cells treated with DMSO. "\*" and "#" indicate significantly different from wt-CFTR treated with DMSO and from respective variant without VX-809, respectively (*p* < 0.05). (**C**) BHK cells expressing wt-, F508del-CFTR alone or jointly with ∆RIL, ∆RES, and ∆RIL-∆RE<sup>S</sup> were subjected to pulse-chase (see Materials and Methods) for the indicated times (0, 0.5, 1, 2, and 3h) before lysis and immunoprecipitation (IP) with the anti-CFTR 596 Ab. After electrophoresis and fluorography, images were analyzed by densitometry. (**D**) Turnover of immature (band B) CFTR for different CFTR variants is shown as the percentage of immature protein at a given time point of chase (P) relative to the amount at t = 0 (P<sup>0</sup> ). (**E**) Efficiency of processing of band B into band C is shown as the percentage of band C at a given time of chase relative to the amount of band B at t = 0. "\*" and "#" indicate statistical significantly different (*p* < 0.05) from wt-CFTR and F508del-CFTR, respectively. Data represent mean ± SEM (*n* = 5).

Given the very significant stabilization of immature ∆RES-F508del-CFTR (Figure 1B), next, we determined how removal of RE<sup>S</sup> affected the processing efficiency and the turnover of the F508deland ∆RIL-F508del-CFTR variants. To this end, we performed pulse-chase experiments (Figure 4C,D) and indeed our results revealed that ∆RE<sup>S</sup> very significantly stabilized immature F508del-CFTR not just relatively to F508del-CFTR but to levels even significantly higher than those of wt-CFTR (Figure 4C,D). Indeed, our data show that although F508del-CFTR without RE did not traffic to the PM, it showed a dramatic stabilization of its immature form (evidencing a turnover rate ~2× lower than that of wt-CFTR. It should be noted that the turnover of F508del-CFTR itself is ~1.4×faster vs. wt-CFTR, thus this stabilization (of ~3× vs. F508del-CFTR) represents indeed a massive stabilization. Interestingly, this stabilizing effect was no longer significant for ∆RIL-∆RES-F508del-CFTR nor for ∆RIL-F508del-CFTR, the latter being equivalent to wt-CFTR (Figure 4C,D). Removal of RI<sup>S</sup> from F508del-CFTR did not stabilize its turnover (data not shown). As to removal of either RE<sup>S</sup> or RI<sup>S</sup> from wt-CFTR, it did not affect the processing efficiency or the turnover vs. wt-CFTR (Figure S2).

In summary, levels of ∆RIL-∆RES-F508del-CFTR at the PM are equivalent to those of wt-CFTR.
