FIGURE 2.
CNK3 positively regulates ENaC surface expression and activity. A, knockdown of endogenous CNK3 decreases ENaC activity in mpkCCDc14 cells. mpkCCDc14 cells stably expressing CNK3 or control (non-target) shRNA were plated on Transwell filters and allowed to grow to high resistance. The cell monolayers were then treated with aldosterone (1 μm for 3 h) prior to determination of ENaC activity. Shown is the -fold change in aldosterone-induced equivalent short circuit current in CNK3 shRNA-expressing cells. Also shown is a representative blot demonstrating knockdown of endogenous CNK3. ***, p < 0.001 compared with control (n = 6). B, cell surface biotinylation assay for ENaC in HEK 293T cells transiently transfected with FLAG-tagged α-, β-, and γ-mENaC subunits. After transfection, cells were treated with EZ-Link sulfo-NHS-biotin before protein extraction and recovered by affinity immobilization using NeutrAvidin-conjugated agarose beads followed by immunoblot (IB) analysis with anti-β-ENaC antibody. Shown is a graphical representation of CNK3-dependent augmentation of surface ENaC based on multiple repeats of biotinylation assays. Also shown is a representative blot demonstrating CNK3 overexpression. ***, p < 0.001 compared with control (n = 4). Error bars represent S.E. WCL, whole-cell lysate.