CAP2 cleaves α-, β-, and γ-ENaC at a conserved basic residue in the pre-M2 region. WT α-, β-, and γ-ENaC–untagged or –double tagged (HA-NT/V5-CT) or α-K561A, β-R503A, and γ-R515A ENaC–tagged subunits (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. Total protein lysates were prepared from oocytes and Western blot analysis was conducted using anti-V5 monoclonal antibodies (A). Black arrows indicate full-length (FL) and cleaved ENaC subunits. Batches of oocytes were extracted from three different frogs. Representative experiments are shown (n = 3). (B) α-, β-, and γ-ENaC human, mouse, and rat pre-M2 region sequences alignment. TMII, transmembrane 2.