Binding of afadin to the αE-catenin–β-catenin complex in solution in vitro. (A) Schematic representation of the domain/region structures and the binding partners of afadin and αE-catenin. The following domains of afadin are shown: RA1, RA2, Ras-associated domain-1 and -2; FHA, forkhead-associated domain; DIL, dilute domain; PDZ, PDZ domain; PR1, PR2, PR3, proline-rich domain-1 to -3; CC, CC region; and FAB, FAB domain. The following domains of αE-catenin are shown: N, N domain; MI, MI subdomain; MII, MII subdomain; MIII, MIII subdomain; and C, C domain. (B) Schematic representation of FL αE-catenin, its mutants, and fusion proteins. (C) Binding of afadin to the αE-catenin–β-catenin complex. Each of the indicated proteins was incubated with the FLAG-tagged fragment of CC-afadin and immunoprecipitated (IP) with the anti-FLAG mAb. The samples were analyzed by SDS-PAGE with CBB staining. Single and double daggers indicate the bands of αE-catenin fragments coimmunoprecipitated with CC-afadin and those of immunoprecipitated CC-afadin, respectively. (D) Quantitative analysis of C. The band intensity of immunoprecipitated MII-C in the presence of CC-afadin is normalized to 1.0.