Fig. 4. P2Y₂R-v integrin interaction regulates Rho-mediated signaling. (A,B) Human 1321N1 cells expressing the WT or RGE mutant P2Y₂R were incubated with the indicated concentration of UTP for 5 minutes. Cell lysates were prepared and analyzed by immunoblotting with (A) anti-phospho-myosin light chain 2 (p-MLC-2) or (B) anti-phospho-cofilin antibodies. (C) Cells expressing the WT P2Y₂R were incubated overnight with or without 10 µg/ml anti-v5 or anti-3 antibody, then stimulated with 100 µM UTP for 5 minutes. Cell lysates were prepared and analyzed by immunoblotting with anti-phospho-cofilin antibodies. (D) Cells expressing the WT P2Y₂R were pretreated at 37°C in serum-free medium with the Rho-dependent kinase inhibitor Y-27632 (10 µM) for 1 hour or 200 ng/ml Bordetella pertussis toxin (PTX) overnight, then stimulated with 100 µM UTP for 5 minutes. Cell lysates were analyzed by immunoblotting with anti-phospho-cofilin antibodies. Protein loading in each lane was evaluated by stripping the membrane of antibodies and re-probing with anti-actin antibodies. Blots representative of 3-5 experiments are shown.