Association of Tara with Trio RhoGEF, which binds E-cadherin. (A, a) Coimmunoprecipitations among YFP-E-cadherin, HA-Trio, and HA-Tara. After the transient expression (see input) in HEK293 cells of tagged forms of Tara, E-cadherin, and Trio, a reported binding partner of Tara, YFP–E-cadherin was detected in the immunoprecipitates of HA-Tara in the presence, but not the absence, of HA-Trio. (b) Linkage between Trio and E-cadherin. HA-Trio was pulled down by a GST fusion protein of the cytoplasmic domain of E-cadherin. (c) Immunofluorescence of transiently expressed HA-Trio. The cell–cell AJs lit up, as indicated by the Tara costaining pattern. Bar, 5 µm. (B) Schematic drawing of Trio RhoGEF and the Tara deletion constructs. (C) Immunofluorescence micrographs of Tara-KD MDCK cells stably transfected with HA-tagged Tara deletion mutants. All the constructs except for the one lacking the mid-domain were localized to the AJ and up-regulated the immunofluorescent signals for E-cadherin. HA, green; E-cadherin, red. Bar, 10 µm. (D) Pull-down assay of full-length and mid-domain Tara, and full-length Tara lacking the mid-domain, with GST fusion proteins of the Trio RhoGEF RhoG/Rac1-GEF domain (GEF-D1) or GST (a control). The mid-domain of Tara was essential for Tara’s interaction with the GEF-D1 domain.