FRET measured by donor dequenching after acceptor photobleaching in HEK293T cells cotransfected with eCFP-STIM1 and eYFP-STIM1.

Part A: CFP-STIM1 images before and after photobleaching of the acceptor within the indicated region (Left column); acceptor YFP-STIM1 intensities before and after photobleaching in the indicated region (Right column). Part B: images acquired near the cell adhesion surface after stimulation of cells with 2 µM TG. Part C: the bar graphs representing FRET efficiency (E) are from 20 independent experiments such as those in part A. The efficiency was determined by the acceptor photobleaching method and was measured only in the (acceptor) bleached area. Cells outside the bleached region were used as controls. Part D: the bar graphs representing FRET efficiency (E) are from the 20 independent experiments in part B. All data are represented as mean±S.D. The significance levels indicated are as follows: **P<0.001. Scale bars, 20 µm.