Effects of ILK overexpression on impaired PAC-1 binding in talin knocked-down parental cells. (A) Dot plot analysis of PAC-1 binding to cotransfectants of empty plasmid, talin-F3, or ILK with shRNA. GFP was used as a transfection marker. An empty plasmid (2 μg), a plasmid encoding a talin-F3 domain (2 μg), or rat ILK (2 μg) was cotransfected with plasmids encoding EGFP (0.5 μg) and shRNA (4 μg) to parental cells. Cells were stained with PAC-1 for flow cytometry. (B) Quantitative estimates of PAC-1 binding to the indicated transfectants. PAC-1 binding was measured in cells that showed high levels of GFP fluorescence (boxed regions in panel A) using flow cytometry. Nonspecific PAC-1 binding was measured in the presence of Ro44-9883, and specific PAC-1 binding was determined by subtracting the MFI of nonspecific binding from the MFI of PAC-1 binding to the indicated transfectants. Data are mean ± SD of 2 experiments.