Retroviral vectors, transgene expression, and function. (A) Bicistronic MSCV-based retroviral vectors expressing EGFP and (HA)3RhoAN19 (▦), or Flag-Rac2D57N (▧). IRES indicates internal ribosome entry site. (B) Immunoblot analysis of transgene expression in the low-density bone marrow transduced cells probed for HA-tag (left), flag-tag (right), or β-actin (loading control). The blots are representative of 3 independent experiments with similar results. (C) Rhothekin/Pak pull-down assays to quantitate active, GTP-bound RhoA (Rho GTP), and Rac (Rac GTP) after specific agonist treatment. The level of β-actin is shown below as loading control. PDGF indicates platelet-derived growth factor; LPA, lysophosphatidic acid. The result is representative of 3 independent experiments.