Fig. 7. (A) Targeting of active ERK1/2 to focal contacts. Confluent PANC-1 cells were incubated overnight in DMEM without supplements. After wounding, cells were treated with DMEM without supplements (a-a"), with 10 µM LPA (b-b"), or with 10 µM LPA and 25 µM PD98059 (c-c"). Cells were fixed after 3 hours and were costained for phosphorylated ERK1/2 (pTpY-ERK1/2 antiserum and Alexa488-conjugated antibodies) and vinculin (anti-vinculin and Cy3-conjugated antibodies). Merged images are shown in a", b" and c". Arrows point to focal contact structures. Microscopy was performed with an inverse fluorescence microscope. Bars, 10 µm. (B) Localization of active ERK1/2 during LPA treatment. Serum-starved PANC-1 cells were wounded, treated for 3 hours, 5 hours and 8 hours with 10 µM LPA, fixed and then stained for phosphorylated ERK1/2 using pTpY-ERK1/2 antiserum and Alexa488-conjugated antibodies. Bars, 20 µm.