CRP1 binds α-actinin along actin stress fibers. REFs were co-transfected with pECFP-CRP1 and pEYFP-α-actinin and FRET microscopy carried out as described in Experimental Procedures. (A) Overlay image of cell expressing CFP-CRP1 and YFP-α-actinin showing the ROI that was photobleached during the FRET experiment. A color scaled image of the CFP fluorescence of the cell prior to (B) and immediately following (C) the photobleaching period. Red represents a high signal and blue, a low signal. Bar = 10 μm. (D) The intensity of the CFP fluorescence within the ROI was quantified for each image captured during the experiment. (E) The distribution of FRET efficiencies for the photobleached ROIs, Ef, and control non-photobleached ROIs, Ec, were plotted on the bar graph.