PECAM-1 engagement by anti-PECAM/NCs induces RhoA activation and mobilization. (A) RhP and ΔCD cells were incubated with anti-PECAM/NCs for 0, 5, and 15 minutes. RhoA activity was then measured using GST-RBD to pull-down active RhoA (GTP-Rho) and blotted with anti-Rho. Total Rho in cell lysates was also determined. (B) Relative RhoA activation was quantified by densitometry and indicated as fold increase of active RhoA in relation to total RhoA. RhP (C) and ΔCD (D) cells transfected to express EGFP-RhoA were incubated for 15 minutes at 37°C with 1 μm diameter, nonfluorescent anti-PECAM/NCs to visualize RhoA redistribution to nanocarrier contact-sites in RhP (Ciii) and ΔCD cells (Diii). After permeabilization, the cells were stained with red Alexa Fluor 594 phalloidin to label filamentous actin (Ci,Di). Phase contrast images (Cii for RhP and Dii for ΔCD) served to locate anti-PECAM/NCs with respect to the cell borders (marked by a dashed line). Lower panels in C and D show 4-fold magnified images of the areas outlined by the insets in RhP (Civ-Cvi) and ΔCD (Div-Dvi) cells.