FRET analyses of BRCA1 and its interacting partners at the PM. (A) Acceptor photobleach FRET on PMA-stimulated HeLa cells. Endogenous BRCA1 was detected using Alexa Fluor 488 (donor); ERM was detected using Cy3 (acceptor). Increase in donor fluorescence was measured after photobleaching of the acceptor. (B) Pre- and postbleach images are shown for a representative ROI (white box in A). (C) Absolute increase in donor fluorescence mean FRET efficiency. 5.79% ± SEM = 0.85 (n = 23). A negative control (Cy3 acceptor targeted to the transferrin receptor) gave a mean FRET efficiency of 1.37 ± SEM = 0.97 (n = 15). P < 0.001 (t test). (D–F) FLIM-based FRET between EGFP-CTD and ERM (detected using Cy3) in untreated HeLa cells. Graph shows the percentage of pixels with a lifetime shortened enough to indicate a FRET efficiency (E) >20% (means ± SEM; n = 5). There is a significant difference (P < 0.05) between EGFP lifetime in the absence of an acceptor (representative cells shown in E) and when ERM-Cy3 is present as an acceptor (representative cells shown in F). A positive control in which the Cy3 acceptor was targeted to EGFP-CTD using an anti-GFP antibody and Cy3 is also shown (anti-GFP). (E and F) Bars, 10 µm.