<p>FRET experiments were conducted in tobacco leaves through transient 35S-driven overexpression of NF-YCs tagged with mCer3 and HY5 or NF-YB2 tagged with YFP. <b>A)</b> Nuclei expressing both mCer3 and YFP constructs were assayed for FRET through both FRAP and FLIM. <b>B)</b> A FRAP curve representative of a positive FRET result between two known interacting proteins, NF-YB2 and NF-YC9. Fluorescence intensity was calculated relative to the pre-photobleached intensity of each fluorescent protein. Yellow bars represent the timing of photobleaching events. <b>C)</b> FLIM was employed to detect FRET through lifetime measurements before and after acceptor photobleaching (FRAP) within the same nucleus. Each point is an independent combination of mCer3- and YFP-tagged proteins, and represents the shift in fluorescent lifetime elicited by acceptor photobleaching. Scale bar in A) represents 5μm. Error bars in B-C) represent 95% confidence intervals with an n ≥ 3.</p
