FIGURE 2.

NHERF3 directly binds to the C terminus of NHE3. Purified His₆ fusion proteins making up the entire NHE3 C terminus (CT) were generated and divided into four parts (F1–4), separated on SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were incubated with purified recombinant human NHERF3 protein and visualized by primary antibody to NHERF3 and exposed to enhanced chemiluminescence. Protein overlay assays demonstrate that purified recombinant human NHERF3 binds to the full-length C-terminal fragment of NHE3 (aa 475–832) and the F2 region of NHE3 (aa 589–667). Lower band in the full-length NHE3 C-terminal fragment is assumed to be a breakdown product containing the F2 domain. Results were obtained from three individual experiments. IB, immunoblot.