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. 2006 Jun;26(11):4327–4338. doi: 10.1128/MCB.02393-05

FIG. 2.

FIG. 2.

Molecular interaction between the AICD and Fe65 factor examined by FRET. (a, b, and c) Quantitative FRET analysis using acceptor bleaching in SK-N-SH cells cotransfected with Fe65 and mock (a), C50 (b), or C50T668A-EGFP (c) expression constructs. Cells were immunostained with anti-Fe65 antibody and visualized with Cy3. Left panels, EGFP signal (mock, C50, or T668A C50) using 488-nm excitation and Cy3 (Fe65) signal using 543-nm excitation before photobleaching and EGFP signal (mock, C50, or T668A C50) using 488-nm excitation and Cy3 (Fe65) signal using 543-nm excitation after photobleaching of the acceptor (Cy3) fluorophore with intense 543-nm laser light (white rectangle). Bar, 10 μm. Middle panels, intensity of the region of interest (ROI), quantification of fluorescence intensity in a representative example also shows a substantial increase in fluorescence after the bleach only in the C50-transfected cells. Right panels, graphs show the percentages of relative fluorescence intensity. Data represent means ± standard errors of results from four separate experiments. **, P < 0.01; *, P < 0.05 (by ANOVA).