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. 2011 Mar 4;286(18):16308–16320. doi: 10.1074/jbc.M110.206672

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Demonstration of FRET between YFP-PLD2 and CFP-Rac2 and stoichiometry of PLD2 and Rac2 binding. COS-7 cells were transfected with CFP-Rac2 (the donor in the FRET pair) and with YFP-PLD2 (the acceptor of the FRET pair) for 48 h. When ready for microscopy, cells were treated with 10 nm EGF for 3 min. Fluorescence imaging showed basic wide field FRET microscopy: I_A (PLD2-YFP), I_D (Rac2-CFP), and I_F (FRET) images. Color panels show calculated FRET images: E_A, E_D, and R_M; color bars, magnitude E_A, E_D, or the ratio of acceptor to donor molecules (R_M). We generated purified, recombinant, tagged proteins (HA-Rac2 and Myc-PLD2WT) from a baculovirus/insect cell expression system.