HGAL interacts with PDZ-RhoGEF through its C-terminal PDZ binding motif. (A) HeLa cells were co-transfected with pcDNA3.1-PDZ-RhoGEF and one of the following plasmids: pcDNA3.1, pcDNA3.1-HGAL, pcDNA3.1-HGALΔPDZ and pcDNA3.1-(1-118)HGAL. Total cellular lysates were prepared and immunoprecipitated with anti-HGAL and anti–PDZ-RhoGEF antibodies and analyzed by Western blotting with anti–PDZ-RhoGEF and anti-HGAL antibodies, as indicated. The corresponding total cellular lysates were subjected to Western blot analysis using anti-HGAL and anti–PDZ-RhoGEF antibodies. (B-C) HeLa cells transfected with pcDNA3.1, pcDNA3.1-HGAL or pcDNA3.1-HGALΔPDZ were used for RhoA pull-down (B) and analysis of luciferase activity from the SRF-driven luciferase reporter construct pSRE-Luc as described in “Luciferase reporter assays” and shown in Figures 1B and 3A. Numbers refer to luciferase activities representing means + SD of the mean of 3 independent experiments, each performed in triplicate. * indicate statistically significant difference (all below P < .01). Results in panels A and B are also representative of 3 independent experiments.