VASP regulates the interaction between β1 and talin. (A) Example FLIM images of β1-GFP–expressing fibroblasts cotransfected with mCherry-talin and either WT-VASP-myc or S153A VASP-myc. Cells were fixed and stained with an anti–myc-Cy5 antibody to detect VASP expression. Cells were then subjected to FRET analysis by FLIM. Images show the GFP multiphoton intensity image and (where appropriate) corresponding wide-field image of the acceptor and myc-tagged VASP. Lifetime images mapping spatial FRET across the cells are depicted using a pseudocolor scale (blue, normal lifetime; red, FRET). Bars, 10 µm. (B) Example FLIM images of β1-GFP–expressing fibroblasts cotransfected with mCherry-talin and either control siRNA (con) or siRNA directed against β3 integrin, RIAM, or VASP. Cells were fixed and subjected to FRET analysis by FLIM. Images show the GFP multiphoton intensity image and corresponding wide-field image of the talin acceptor. Lifetime images mapping spatial FRET across the cells are depicted using a pseudocolor scale (blue, normal lifetime; red, FRET). (C) Quantification of FRET efficiency from FLIM data in A and B. The graphs represent mean FRET efficiency of >12 cells of each type over three independent experiments. Error bars indicate ± SEM. *, P < 0.05; **, P < 0.01.