Cooperative effects of HGAL, PDZ-RhoGEF and LARG on RhoA activation and lymphocyte motility. (A) Raji cells transfected with either control siRNA or siRNA for HGAL alone or in combination with siRNAs for PDZ-RhoGEF, LARG or both were seeded on fibronectin. Cellular lysates were prepared and used for RhoA pull-down assay and Western blotting with anti-HGAL, PDZ-RhoGEF, LARG and actin antibodies. Densitometry analysis of normalized RhoA-GTP to total RhoA from 3 independent Western blot experiments is presented. The values in the control specimens were arbitrarily defined as 1. Error bars represent SD. (B) HeLa cells stably transfected with pcDNA3.1 or pcDNA3.1-HGAL were transfected with siRNAs for PDZ-RhoGEF, LARG or both and were seeded on fibronectin. Cellular lysates were prepared and used for RhoA pull-down assay and Western blotting with anti-HGAL, PDZ-RhoGEF, LARG and actin antibodies. Densitometry analysis of normalized RhoA-GTP to total RhoA from 3 independent Western blot experiments is presented. The values in the control specimens were arbitrarily defined as 1. Error bars represent SD. (C) VAL lymphoma cells were transfected with control siRNA or siRNA for HGAL and siRNAs for PDZ-RhoGEF, LARG or both. 48 hours after siRNA transfection, the cells were used for IL-6 and SDF1 chemotaxis assay performed in triplicate, as described in the “Chemotaxis assays.” Means + SD of the mean of 3 independent experiments are demonstrated. Statistically significant differences with *P < .05 and **P < .01.