Distinct associations of filamin with inactive and active integrin αIIbβ3 cytoplasmic face. Representative images of FRET efficiency using acceptor (mVenus) photobleaching of mTurquoise-mVenus pair transfected in CHO-K1 cells. Rings outlining the cell membrane were selected as the region of interest. Intensities within the region of interest before and after bleach were counted using Image Pro Plus 7 and used for calculating FRET efficiency (FRET effi.). Ten cells in each group were analyzed (see supplemental Figure 2A-F). A separation >100 Å between mTur and mVen would abolish FRET. (A) FRET of αIIb-mTur/β3-mVen pair without PMA treatment (left) shows good FRET efficiency, consistent with the association of αIIb/β3 CTs. Upon PMA treatment (right), the FRET efficiency is reduced, consistent with the separation of αIIb/β3 CTs upon integrin activation. (B) FRET of β3-mTur/FLNa Ig21-mVen pair without PMA treatment (left) shows good FRET efficiency, consistent with filamin association with β3 CT in the resting state of integrin. Upon PMA treatment (right), the FRET efficiency is reduced indicating the dissociation of filamin from β3 CT. (C) FRET of αIIb-mTur/FLNa Ig21-mVen pair with (right) and without (left) PMA treatment shows similar FRET efficiency, indicating that filamin remains associated with αIIb CT in both resting and active states of αIIbβ3. (D) Bar plot for FRET measured for αIIb-mTur/β3-mVen, or PC-αIIb/β3-mTur/FLNa Ig21-mVen, αIIb-mTur/PC-β3/FLNa Ig21-mVen cotransfected in CHO-K1 cells with or without PMA treatment. Data are average values ± SEM for 10 cells. ∗∗∗P < .001; ∗P > .05. SEM, standard error of mean; ns, nonsignificant.