The FH1 domain of hDia-1 is critical for binding to the RAGE cytoplasmic
domain. A, schematic diagram of Myc-tagged hDia-1 and truncation
mutants used in this study. Numbers indicate amino acid positions in human
Dia-1. Rho-BD, Rho GTPase binding domain; DID, diaphanous
inhibitory domain; DD, dimerization domain; CC, coiled-coil region;
FH1 and -2, formin-homology region 1 and 2; DAD,
diaphanous autoinhibitory domain. B, to analyze the domain(s) of
Dia-1, which interacts with the RAGE cytoplasmic domain, coimmunoprecipitation
(IP) experiments were performed. SK-BR-3 cells were transiently
cotransfected with the indicated Myc-tagged hDia-1 constructs and the
His-tagged RAGE cytoplasmic domain. Immunoprecipitation was performed with
anti-His antibodies followed by Western blotting (WB) with anti-Myc
antibodies (left panel). Western blot analysis was performed on input
lysate as indicated with anti-Myc and anti-His antibodies for hDia-1 and RAGE
cytoplasmic domain constructs, respectively (right panel). Note that
the hDia-RhoBD does not bind to the RAGE cytoplasmic domain. All panels
(A-B) are representative images of multiple independent
experiments.