Cleavage of α-, β-, and γ-ENaC–tagged subunits by WT but not inactive CAP2 S387A in oocytes. WT α-, β-, and γ-ENaC–double tagged (HA-NT/V5-CT) and α-, β-, or γ-ENaC–untagged subunits (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. Total protein lysates were prepared from oocytes, and Western blots analysis were conducted using anti-HA (A) and anti-V5 (B) monoclonal antibodies. Western blots of surface biotinylated and total pools of α-ENaC HA-NT (C) and γ-ENaC V5-CT (D) fragments caused by WT but not inactive CAP2 (CAP2 S387A). Actin expression was detected with an anti-actin monoclonal antibody as control (D). A β-ENaC 90-kD HA-NT fragment was detected at the surface pool (not depicted). NS, nonspecific. Batches of oocytes were extracted from three different frogs. Representative experiments are shown (n = 3). (E) Linear diagram of ENaC residues cleaved by CAP2. NH2, amino terminus; COOH, carboxy terminus; TM1, transmembrane domain 1; TM2, transmembrane domain 2.