Establishment of mutant cells expressing inactivated αIIbα6Bβ3. (A) FACS sorting of mutant cells. CHO cells expressing constitutively activated αIIbα6Bβ3 (parental cells) were treated with a chemical mutagen EMS for 20 hours. After cell culture for 7 days, the cells were incubated with an activation-specific mAb against αIIbβ3, PAC-1 (conjugated with PE), and a β3-specific mAb Y2/51 (conjugated with fluorescein isothiocyanate). The cells that showed high levels of Y2/51 binding but did not bind PAC-1 were sorted out by a FACS. These cells were expanded and resorted, and then the mutant cells were cloned. (B) Characterization of mutant clones. Clones defective in PAC-1 binding were confirmed by flow cytometry (i), and the clones were examined for a phenotypic restoration of PAC-1 binding by DTT treatment (ii,iii). Nonspecific binding was shown by cells incubated with the secondary Ab alone (i). Mutant cells were treated with DTT or buffer and washed once, and then the cells were incubated with PAC-1 (ii,iii). (C) Flow cytometry showing expression of αIIbα6Bβ3 on parental or mutant cells. Cells were incubated with mAbs against αIIb (SZ22), β3 (Y2/51), and αIIbβ3 (HIP8). After washing, bound mAbs were detected with an AlexaFluor 488–conjugated secondary Ab. Nonspecific binding is shown by the cells stained with the secondary Ab alone (thin solid line). Results are a representative of 3 independent experiments. (D) Immunoblotting analysis of talin in the parental and mutant cells. Whole-cell lysates were electrophoresed, transferred to a polyvinylidene difluoride membrane, incubated with antitalin mAb or anti-GAPDH polyclonal Ab, and then detected by chemiluminescence.