Figure 1.
(A) FLAG-epitope placement into the rat ENaC sequence. The alignment of the extracellular loop sequences for the rat, human, and Xenopus ENaC subunits demonstrated the most important divergence in the region near the first transmembrane domain M1. This region was chosen to insert (α and γ subunits) or to replace (β subunit) the FLAG sequence (DYKDDDDK) at the indicated positions. (B) Immunoprecipitation of the FLAG-tagged ENaC subunits, using M2Ab*. Oocytes were injected with an α, β, or γ nontagged subunit alone (lane 1), or with an α, β, or γ FLAG-tagged subunit alone (lanes 2 and 3). Oocytes were metabolically labeled with [35S]methionine for a 4-h pulse followed by a 4-h chase period. Membranes were prepared, and proteins were immunoprecipitated and analyzed on 5–8% gradient SDS/PAGE. Immunoprecipitations of the samples corresponding to lane 3 were performed in the presence of 1000-fold molar FLAG peptide excess over the antibody concentration.