Fig. 1. P2Y₂R-mediated Rho activation and stress fiber formation require P2Y₂R interaction with v integrins. (A) Cell surface expression of HA-tagged wild-type (WT) or RGE mutant (RGE) P2Y₂Rs in 1321N1 cells was determined by flow cytometry. Cells stained with only secondary antibody were used as a negative control. (B) Cells expressing the WT or RGE mutant P2Y₂R were treated with or without 1 mM UTP for 5 minutes prior to measuring ERK1/2 phosphorylation and Rho activity. Results shown are representative of three experiments. (C) Cells expressing the WT or RGE mutant P2Y₂R were treated with or without 1 mM UTP or 20% FBS (positive control) for the indicated time and stress fibers were visualized by staining filamentous actin (shown in green) with Oregon-Green-labeled phalloidin. Texas-Red-labeled DNase I was used to label monomeric actin (shown in red). More than 100 cells were examined in three separate experiments, and representative images are shown. Bar, 50 µm.