FIGURE 2.
Expression of ENaCs and isolation from tsA 201 cells. A, cells were transiently transfected with DNA encoding HA/V5-tagged α-, β-, or γ-ENaC. Cells were fixed, permeabilized, and incubated with either mouse monoclonal anti-HA or mouse monoclonal anti-V5 antibodies, followed by Cy3-conjugated goat anti-mouse secondary antibody. Cells were imaged by confocal laser scanning microscopy. Scale bar, 50 μm. B, cells were singly transfected with αHA/FLAG-ENaC, βHA/His6-ENaC, or γHA/V5-ENaC. Cells were fixed, permeabilized, and incubated with mouse monoclonal anti-FLAG, rabbit polyclonal anti-His6, or mouse monoclonal anti-V5 antibodies, as appropriate, followed by Cy3-conjugated goat secondary antibodies. C, detection of HA/V5-tagged α-, β-, and γ-ENaC in samples isolated from singly transfected cells by anti-HA affinity chromatography. Samples were analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody, followed by horseradish peroxidase-conjugated goat secondary antibodies, with enhanced chemiluminescence detection. Arrowheads indicate molecular mass markers (kDa). D, detection of αHA/FLAG-ENaC, βHA/His6-ENaC, and γHA/V5-ENaC in samples isolated from triply transfected cells by anti-FLAG affinity chromatography. α-, β-, and γ-ENaC were visualized using anti-FLAG, anti-His6, and anti-V5 antibodies, respectively.