ADAP associates with talin-αIIb/β3 complexes in nonhematopoietic (CHO) cells. (A-B) Chimeric proteins VN-talin and αIIb-VC26 that yield BiFC signals in the 488 channel (green) when the proteins are close were inducibly expressed in conjunction with wild-type β3 (VN-talin/αIIb-VC β3) (A) or the truncated β3Δ724 (VN-talin/αIIb-VC β3Δ724) (B) lacking talin-binding sites. After transient transfection of ADAP, harvested cells were plated on fibrinogen without (top) or with (bottom) SFLLRN stimulation, processed as in Figure 2, and stained with antibodies against ADAP (red) and αIIbβ3 (blue) and with Hoechst (magenta) for nuclei. Arrows indicate co-localization of ADAP and BiFC signals to regions of αIIbβ3 staining at the cell periphery and in focal adhesion structures (arrows). Little BiFC signal was observed in VN-talin/αIIb-VC β3Δ724 cells (B). Neither ADAP nor αIIbβ3 staining was observed when control IgGs were used as secondary reagents (C). Results are representative of 3 experiments. (D-E) Quantification of co-localization of ADAP with BiFC signal. (D) Average Pearson correlation coefficient. (E) Quantification of the voxel ratio, an index of the relative fluorescence (eg, red/green) in a unit voxel (n = 4; *P < .05, **P < .01). Results in (D) and (E) represent means ± SEM.
