DAAM1 interacts with the CCC. (A) DAAM1 localizes at cell contacts via its N-terminal region. (Top) Schematic representation of the deletion mutants of DAAM1. (Bottom) HA-tagged full-length or deletion mutants of DAAM1 were transiently expressed in EpH4 cells and immunostained for HA and α-catenin. (B) Interaction of DAAM1 with the endogenous CCC. Lysates of HA-tagged DAAM1-expressing or parental EpH4 cells were subjected to immunoprecipitation with anti-HA antibody, followed by immunoblotting with antibodies against E-cadherin, α-catenin, β-catenin, or HA. (C) Interaction of DAAM1 with E-cadherin. HEK293T cells were transfected with FLAG-tagged E-cadherin with or without HA-tagged DAAM1. Cell lysates were subjected to immunoprecipitation using anti-HA antibody, followed by immunoblotting with antibodies against FLAG or HA. (D) Interaction between endogenous DAAM1 and E-cadherin. EpH4 cell lysates were subjected to immunoprecipitation by anti-DAAM1 antibody or normal rabbit IgG, followed by immunoblotting with anti–E-cadherin antibody. (E) Interaction of DAAM1-N with the cytoplasmic region of E-cadherin (E-cad–cyto). Lysates of EpH4 cells expressing DAAM1-N were incubated with purified GST-tagged proteins as indicated and subjected to pull-down assays. Note that only GST–E-cad–cyto specifically interacted with DAAM1-N. (F) Distribution of DAAM1 to cell junctions depends on E-cadherin. EpH4 cells were treated with an E-cadherin–specific siRNA for 2 d and then immunostained. Arrows indicate Nectin-1α–positive cell–cell contacts in E-cadherin–depleted cells. (A and F) Bars, 10 µm.