Effects of RhoGDIβ on Rac1 expression and subcellular localization of RhoGTPases. (A) RhoGDIβ, RhoGDIβ, Rho, CDC42, and Rac1 were detected by western blotting of cell lysates from H9c2 cells stably expressing RhoGDIβ. (B) Total RNA was isolated from H9c2 cells stably or transiently expressing RhoGDIβ. Rac1 transcripts were analyzed by northern blotting. The lower EtBr panels of (B) represent the 28S and 18S loading controls. (C) Membrane (Mem.) and cytosolic (Cyto.) fractions from H9c2 control (C) and RhoGDIβ-expressing cells were analyzed by immunoblotting for Rac1, Cdc42, and RhoGDIβ. (D) GTP loading in Rac1 was determined utilized PAK PBD binding assay in H9c2 cells stably expressing RhoGDIβ.