Analysis of ILK in mutant cells. (A) Immunoblotting using anti-ILK Ab. Whole-cell lysates obtained from CHO cells, parental cells, mutant cells, and mutant cells transiently transfected with rat ILK cDNA were electrophoresed on SDS-PAGE and immunoblotted with rabbit anti-ILK polyclonal or rabbit anti-GAPDH polyclonal Ab. (B) Flow cytometry showing expression of activated β1 integrins. Cells were incubated with mAb specific for hamster β1 (7E2) or activated β1 (9EG7). Bound mAbs were detected with PE-conjugated secondary Ab. A thin solid line represents nonspecific binding to cells stained with the secondary Ab alone. Results are representative of 3 independent experiments. The mean fluorescence intensities of 7E2 and 9EG7 binding are indicated on the histograms. (C) Effects of ILK expression on β1 integrin activation. Mutant cells were transiently transfected with GFP-fused wild-type ILK (GFPILK-WT) cDNA or GFP cDNA. After 72 hours, 9EG7 binding to transfectants was analyzed by flow cytometry. 9EG7 binding was expressed as binding normalized to β1 expression (7E2 binding). Data are mean plus or minus SD of 3 independent experiments. (D) Specific binding of PAC-1 Fab fragment to parental cells and mutant cells. Nonspecific binding of PAC-1 Fab fragment to cells was measured in the presence of Ro44-9883, and specific PAC-1 Fab binding was estimated by subtracting the MFI of nonspecific binding from the MFI of PAC-1 Fab binding to the indicated cells. Data are mean plus or minus SD of 3 experiments, and the calculated values are displayed on the graph. (E) The activation index of ILK-transfected mutant cells. The activation index using PAC-1 Fab was calculated by the formula shown in “Flow cytometry.” A value of 100% represents maximal PAC-1 Fab binding to the DTT-treated cells. Data are mean plus or minus SD of 3 independent experiments, and the values are displayed on the graph.