Figure 4.

N‐myc downregulated gene 1 differentially regulates activities of Rho GTPases. A, Detergent‐solubilized extracts from HEK293 and DU145 were incubated with immobilized GST‐RBD beads, to pull down active RhoA, followed by immunoblotting with a RhoA antibody. B, Detergent‐solubilized extracts from HEK293 and DU145 were incubated with immobilized GST‐PBD beads, to pull down active Rac1 and Cdc42, followed by immunoblotting with a Rac1 antibody. C, Detergent‐solubilized extracts from HEK293 and DU145 were incubated with immobilized GST‐PBD beads, to pull down active Rac1 and Cdc42, followed by immunoblotting with a Cdc42 antibody. Each experiment was repeated three times and the amounts of GTP‐RhoA/Rac1/CDC42 were analyzed by densitometer. Left panel in A, B, and C shows representative blot of the GTP‐RhoA/Rac1/CDC42 activity assay. Right panel shows quantification and statistical analysis of the GTP‐RhoA/Rac1/CDC42. ^∗p < 0.05. (D) Representative confocal images for immunofluorescence staining of NDRG1‐knockdown (LNCaP and DU145) and control cells stained with vinculin (green) and rhodamine‐conjugated phalloidin (red) to stain the focal adhesion and actin stress fibers, respectively. Magnified view of LNCaP cells shown in lower panel. Insets are inverted images for better visualization of filopodial extensions (arrow heads). NDRG1 knockdown increases filopodial extensions. (E) Quantification of filopodial numbers in NDRG1‐knockdown and parental cell lines.