VASP-RIAM binding is enhanced in the absence of β3. (A, right) Confocal images of β3-GFP cells transfected with WT, S153A, or S153D VASP-mCherry fixed and stained for active β1 (9EG7–Alexa Fluor 568, blue). Merged images are shown. Bars, 10 µm. (A, left) The histogram shows Pearson's correlation coefficient analysis of colocalization between VASP and 9EG7. Error bars indicate ± SEM, n = 12 for each. **, P < 0.01. (B) WT, β3−/−, or β3-GFP cells were lysed and subjected to immunoprecipitation with anti-VASP or control antibodies. Bound and input lysates were Western blotted for RIAM or VASP. Blots are representative of similar results from three independent experiments. (C, left) Example images of WT and β3−/− cells transfected with GFP-VASP alone (left, control) or in combination with mCherry-RIAM (right) subjected to FRET analysis by FLIM. Images show the GFP multiphoton intensity image and (where appropriate) corresponding wide-field image of the acceptor. Lifetime images mapping spatial FRET in cells are depicted using a pseudocolor scale (blue, normal lifetime; red, FRET). (C, right) Histogram of quantification of FRET efficiency from FLIM data. The graph represents mean FRET efficiency of >14 cells of each type over four independent experiments. Error bars indicate ± SEM. **, P < 0.01. (D) Example FRET images for WT and β3−/− cells transfected with GFP-RIAM and mCherry-talin. Quantification of FRET efficiency from FLIM data as in C. The graph represents mean FRET efficiency of >12 cells of each type over three independent experiments. Error bars indicate ± SEM. *, P < 0.05. Bars, 10 µm.