3C). Figure 3. G22-A39 interacts specifically with the ��-ENaC subunit. A) Typical Western blot of the triple-transfected ���¦�-ENaC peptide pulldown assay. The pulldown assay customer review was performed with 1 V5-tagged subunit and 2 untagged subunits … ��-ENaC glycosylation is required for the ��-ENaC/G22-A39 interaction The predicted molecular mass of a nonglycosylated ��-ENaC subunit is ~73 kDa. However, the extracellular loop of ��-ENaC contains 12 possible sites for N-linked glycosylation, and ��-ENaC is typically observed at 94�C96 kDa due to extensive N-linked glycosylation (10). As seen in Fig. 3, the molecular mass of the ��-ENaC subunit, which predominantly interacted with the G22-A39 peptide, was ~94 kDa. To confirm that this was glycosylated ��-ENaC, the elutions of the G22-A39/ ��(V5��)�� ENaC pulldowns were deglycosylated with EndoH (Fig.
4). On treatment with EndoH, the 94-kDa ��-ENaC band shifted to ~73 kDa, consistent with the deglycosylation of ��-ENaC (Fig. 4A). This experiment was repeated with the ��-ENaC subunit expressed alone. On EndoH treatment, this band shifted to ~73 kDa (Fig. 4B). EndoH treatment could only be performed once the pulldown assay had been completed; therefore, to test whether G22-A39 could interact with nonglycosylated ��-ENaC, we exposed cells to tunicamycin, an inhibitor of N-linked glycosylation (32). The pulldown assay was then performed on the tunicamycin-treated cell lysate (Fig. 4C). As seen in the input lane, treatment with tunicamycin reduced the molecular mass of the ��-ENaC subunit to ~73 kDa, confirming that the protein was deglycosylated.
No ��-ENaC was observed in the elution of the tunicamycin-treated pulldown. Combined with the EndoH data, this indicates that G22-A39 is interacting with a specific, glycosylated form of ��-ENaC. Figure 4. ��-ENaC/G22-A39 interaction is glycosylation dependent. A) Typical Western blot of the ���¦�-ENaC peptide pulldown assay with the ��-ENaC subunit V5-tagged and untagged ��- and ��-ENaC subunits. Pulldown … G22-A39 attenuates ASL hyperabsorption in CF HBECs through ENaC inhibition To determine whether G22-A39 could inhibit ENaC-dependent ASL absorption in native airway epithelia, we measured ASL height over time in both NL and CF HBECs after treatment with G22-A39 (Fig. 5A, B). SPLUNC1 is endogenously secreted by both NL and CF HBECs, which could affect ASL volume regulation, especially in NL HBECs (16).
Thus, we standardized the mucosal washing/volume-loading protocol accordingly so that every culture had endogenous SPLUNC1 removed prior to t = 0. Each culture was left with undisturbed ASL for 24 h. They were then incubated for 30 min with 500 ��l PBS, followed by 2 quick successive washes Batimastat with 500 ��l PBS. Then, 20 ��l of PBS containing rhodamine-dextran was added as a volume challenge with or without peptide.