Fig. 3.

Immunoprecipitated Western blot (IP WB) confirming endogenous Xenopus ENaC β-subunit associates with phosphatidic acid, phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidylinositol 3,4,5-trisphosphate (PIP₃) in 2F3 cells. Cell lysate from Xenopus 2F3 cells was incubated in the presence of beads conjugated to either phosphatidic acid, PIP₂, or PIP₃. The complex of conjugated beads and interacting proteins were subject to a series of washes before being eluted in sample buffer. Eluted proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes before being blocked and probed for Xenopus ENaC β-subunit using ENaC-β 60 antibody. As a control, unconjugated beads were used to determine nonspecific binding. WCL represents whole cell lysate.