PDZ-RhoGEF interacts with HGAL through DH domain. (A) Structure of wild-type and 6 different V5 epitope tagged truncated mutants of PDZ-RhoGEF that were used to transfect Raji lymphoma cells expressing endogenous HGAL (B) and HEK-293T cells cotransfected with pcDNA3.1-HGAL vector (C). PDZ: PDZ domain; RGS: regulators of G protein signaling domain, B: protein sequence binding to actin, DH: Dbl-homology domain, PH: pleckstrin-homology domain. (B-C) Total cellular extracts prepared from the transfected Raji (B) and HEK-293T (C) cells were immunoprecipitated with anti-HGAL and anti-V5 antibodies and analyzed by Western blotting with anti-V5 and anti-HGAL antibodies, as indicated. The corresponding total cellular lysates were subjected to Western blot analysis using anti-HGAL and anti-V5 antibodies. (D) The purified GST-(597-1080)PDZ-RhoGEF or GST proteins were incubated with purified TRX-HGAL or TRX proteins for 12 hours. The co-precipitated HGAL and (597-1080)PDZ-RhoGEF were detected by immunoprecipitation with anti-HGAL and anti-GST antibodies followed by Western blotting with anti-GST and anti-HGAL antibodies, as indicated. Results are representative of 3 independent experiments.