Structural requirements in talin and β3 for talin recruitment and inside-out αIIbβ3 activation in living cells. (A) Effects of talin mutation on BiFC and PAC-1 binding. VN-talin (either wild-type, L325R, or W359A talin) was cotransfected with Rap1a (V12) into CHO cells expressing αIIb-VCβ3 or αIIb-VCβ3 (Δ724). As a control, cells were transfected with Rap1a (V12) but not VN-talin (white bars). 48 h later, BiFC fluorescence, PAC-1 binding, and talin expression in transfected cells were quantified by flow cytometry as described in Materials and methods. Responses are expressed as fold increase compared with cells not transfected with VN-talin. (B) Effects of β3 mutation on BiFC and PAC-1 binding. VN-talin was cotransfected with Rap1a (V12) into CHO cells expressing αIIb-VCβ3, αIIb-VCβ3 (Y747A), or αIIb-VCβ3 (Δ724). As a control, cells were transfected with empty vector (mock). 48 h later, BiFC fluorescence, PAC-1 binding, and talin expression in transfected cells were quantified by flow cytometry. Responses are expressed as fold increase compared with mock controls. (A and B) Data are means ± SEM (error bars) of four experiments.