Effects of ILK siRNA on activated αIIbα6Bβ3 in parental cells. (A) Flow cytometric analysis of PAC-1 binding to siRNA transfectants. Parental cells were transiently transfected with scrambled siRNAs, ILK-specific siRNAs (Ilk467 and Ilk1255), or talin-specific siRNA (Tln5465) at a final concentration of 100 nM. After 72 hours, PAC-1 binding to transfectants was analyzed by flow cytometry in either DTT-treated or nontreated condition. The thin line histogram represents cells incubated with a PE-conjugated secondary Ab alone. Results are representative of 3 independent experiments. (B) The activation index of transfectants. The activation index was determined by the formula shown in “Flow cytometry.” Data are mean plus or minus SD of 3 experiments. (C) Immunoblotting of ILK and talin showing knockdown effects of siRNA transfection. Whole-cell lysates from the indicated transfectants were electrophoresed on SDS-PAGE and immunoblotted with rabbit anti-ILK polyclonal Ab, mouse antitalin mAb 8D4, and rabbit anti-GAPDH polyclonal Ab. (D) Adhesion of siRNA transfected cells or ILK-transfected cells to immobilized fibrinogen. (i) Parental cells were transiently transfected with scrambled Ilk1255 or Ilk1255 at 100 nM. After 72 hours, cell adhesion to microtiter wells coated with the indicated concentrations of fibrinogen was tested in triplicate. (ii) Mutant cells were transfected with an empty plasmid (2 μg) or a plasmid encoding rat ILK (2 μg), and cell adhesion was tested. Results are representative of 3 independent experiments. Data are mean plus or minus SD of triplicate measurements of optical density at 405 nm.