HGAL interacts with PDZ-RhoGEF and LARG and stimulates guanidine exchange activity. (A) Cellular lysates were extracted from unmanipulated isolated GC B lymphocytes and HeLa cells stably transfected with pcDNA3.1-HGAL, Raji and VAL cells seeded on fibronectin and subjected to immunoprecipitation with anti–PDZ-RhoGEF and LARG or HGAL antibodies, as well as control antibodies, followed by anti-HGAL and anti–PDZ-RhoGEF or anti-LARG Western immunoblotting, respectively. (B-C) Raji (B) and HeLa cells stably transfected with pcDNA3.1-HGAL (C) were seeded on fibronectin for 90 minutes and stained with DAPI (blue nuclear staining), antibodies to HGAL (green) and PDZ-RhoGEF or LARG (red). Slides were viewed on a Carl Zeiss LSM510/UV confocal microscope with 63×/1.4 NA plan apochromat objective lens and 2× zoom and processed with Zeiss LSM510 AIM 3.2 SP2 confocal microscope software. Z stack linescan images demonstrating proteins colocalization at the cell membrane (arrow) are shown; higher resolution images are shown in supplemental Figure 6. (D) PDZ-RhoGEF was immunoprecipitated from Raji cells and used in the RhoA N-methylanthraniloyl exchange assay, either alone or with purified HGAL protein. Purified Dbs protein served as a positive control, while immunoprecipitates with beads only, purified HGAL alone and water were used as negative controls. Results are representative of 3 independent experiments.