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English: Fluorescence resonance energy transfer analysis of NtWRKY12 interacting with NtWRKY12, TGA2.1, and TGA2.2. Arabidopsis protoplasts were cotransfected with the indicated expression constructs. (A) Protoplasts transfected with a plasmid encoding a YFP:CFP tandem fusion (solid line) and protoplasts transfected with plasmids expressing unfused CFP and YFP (stippled line) were used as positive and negative FRET controls, respectively. (B–D) FRET data from protoplasts transfected with a combination of NtWRKY12:YFP (B), TGA2.1:YFP (C) or TGA2.2:YFP (D), and NtWRKY12:CFP (solid lines), compared to unfused CFP (stippled lines). After excitation at 457 nm, emission energies were measured in a total of 30, 5 nm wide intervals between 468 and 587 nm using confocal microscopy. Data from five protoplasts were averaged and normalized. FRET is presented by the slopes of lines connecting emission intensities at 475 nm (CFP quenching) and 527 nm (YFP emission). Error bars represent the SEM.