Cytohesin-1 controls the activity of RhoA in several cell types. RhoA is an important regulator of beta-2 integrin activation epitope expression and adhesion of DCs. Using a commercial enzyme linked assay (ELISA), we could show that in Mo-DCs the activity (GTP loading) of the small GTPase RhoA is strongly reduced in cytohesin-1 knockdown cells, compared with control cells. As a positive control for the assay, RhoA activity of RhoA knockdown Mo-DCs was analyzed, which also shows the expected reduction of RhoA activity. The same system was used to measure Rac activity, where RNAi of cytohesin-1 results in an increase of GTP loading (A). Consistently, RhoA activity of RhoA knockdown BM-DCs was also found reduced (B). Overexpression of wild-type cytohesin-1 in EGF-stimulated (50 nM, 1 hour) HeLa cells results in an increase of RhoA activity, compared with the vector control. In contrast, overexpression of the cytohesin-1 E157K mutant results in reduced RhoA activity (C). Cytohesin-1 knockdown down-regulates RhoA activity, but up-regulates GTP loading of Rac1 in EGF-stimulated HeLa (50 nM; 1 hour) cells, as detected by effector protein pull-down and Western blot analysis (panel D bottom panels). Knockdown of RhoA specifically reduces expression of the CD18 327C epitope in Mo-DCs (E) or adhesion under flow of BM-DCs to brain endothelioma (B end5) cells (F). Overexpression of wild-type RhoA but not of the GTP-loading mutant T19N increases static adhesion of GFP-cotransfected Mo-DCs to ICAM-1-Fc (G). In the static adhesion assay, Mo-DCs were stimulated with 50 ng/mL PMA for 60 minutes at 37°C (G). Error bars indicate ± SD. ***P < .001, **P < .01, *P < .05. Each experiment was repeated at least 3 times independently. Each single experiment was performed in duplicate.