The TTT motif in the β2 integrin tail mediates kindlin-3 binding. (A) The interaction between human WT β2 integrin (TTT, left) or mutant β2 integrin (AAA, right) with human kindlin-3 was measured in transfected HEK293 cells using FRET. In each case, representative graphs are shown with 3 measurements before and 3 measurements after acceptor photo-bleaching. Error bars indicate standard deviation. (B) FRET results of the β2–kindlin-3 interaction are summarized as percentage FRET efficiency (N = 23 cells, mean ± SEM). (C) The interaction of talin with WT-β2-integrin or TTT/AAA β2-integrin was assessed by GST pulldowns with GST alone, GST-WT-β2-integrin (WT), and GST-TTT/AAA-β2-integrin (TTT/AAA) from T-cell lysates followed by immunoblotting with an antitalin antibody. Lys, T cell lysate. Experiment is representative of N = 2.